COMPOSITIONS AND METHODS OF USING THERAPEUTIC p53 PEPTIDES AND ANALOGUES

ABSTRACT

The present invention relates to compositions useful in inhibiting Bcl-XL or MCL-1 and disrupting p53-MDM2 and p53-MDMX interactions, and methods of using those compositions for treating a subject for conditions responsive to increasing p53 mediated activity or promoting p53 independent apoptosis, such as treating cancer. In some aspects, the compositions of this invention relate to fusion polypeptides comprising a human serum polypeptide and a p53-peptide, which can be, in some aspects, a p53 derived peptide and/or a p53 activating peptide.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/948,010, filed Nov. 20, 2015, which claims the benefit of priority toU.S. Provisional Application No. 62/083,010, filed on Nov. 21, 2014,which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 20, 2018, isnamed B3497-00208_DIV_Sequence_Listing.txt and is 22,207 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compositions useful in inhibitingBcl-XL or MCL-1 and disrupting p53-MDM2 and p53-MDMX interactions, andmethods of using those compositions for treating a subject forconditions, such as neoplastic disorders, including cancer, that areresponsive to increasing p53-mediated cellular activities or promotingp53-independent apoptosis. In some aspects, the compositions of thisinvention relate to fusion polypeptides comprising a transporterpolypeptide, such as human serum albumin, and a p53-agonist, which canbe, in some aspects, a p53 peptide such as a p53 derived peptide and/ora p53 activating peptide, or a small molecule agonist.

BACKGROUND

Cancer is the general name for a group of more than 100 diseases, whichare diagnosed in more than one million new patients in the United Stateseach year. Although there are many kinds of cancer, they share a commonfeature: abnormal cancer cells either do not undergo efficientapoptosis, and/or are not effectively killed by the patient's immunesystem, leading to the uncontrolled growth of the abnormal cancer cells.Untreated cancers can cause serious illness and death. Althoughidentification of risk factors, early detection, diagnosis, andtreatment options have improved prognosis of many kinds of cancers,cancer remains a leading cause of death.

The p53 tumor suppressor protein plays a critical role in generatingcellular responses to a number of stress signals, including DNA damage,aberrant proliferative signals due to oncogene activation, and hypoxia.Upon activation, p53 is stabilized and moves to the nucleus, where itbinds to DNA in a sequence specific manner and promotes transcriptionalregulation of genes involved in DNA repair, cell-cycle arrest,senescence, and apoptosis. While it is estimated that the p53 gene ismutated in 50% of tumors, increasing evidence reveals that a largepercentage of tumors retain wild type p53, but possess other alterationsin the p53 pathway, which prevents its critical tumor-suppressivefunction.

SUMMARY

In one aspect, the current invention relates to fusion polypeptidescomprising a polypeptide, and a p53 agonist, e.g., a p53 peptide orsmall molecule agonist, for example, comprising a human serum albumin(HSA) polypeptide and a p53 peptide. In some aspects, p53 peptides ofthis invention are p53 derived peptides or p53 activating peptides,including analogs thereof. The p53 agonists of this invention are smallmolecule analogs of the p53 peptides, capable of inhibiting two or moretargets from two essential cellular pathways involved in modulatingapoptosis. In some aspects, the transporter polypeptide may be anynatural or artificial polypeptide, including but not limited to, forexample, animal serum albumin, including but not limited to a humanserum albumin (HSA), or a fragment thereof, animal serum globulin,including but not limited to, an immunoglobulin, (an antibody) orantibody fragment polypeptide, such as an Ig-FC polypeptide, atransferrin polypeptide, an antennapedia peptide, cationic cellpenetrating peptide (TAT), transportan and polyarginine. The fusionpolypeptides of this invention have been shown to be transported intocells, and to surprisingly bind and interact with more than two targetproteins involved in mediating apoptosis, including BCL-XL, MCL, MDM2,and MDMX. In some aspects, the fusion polypeptides inhibit BCL-XL andMCL and MDM2 and/or MDMX, for example, the fusion polypeptides mayinhibit one or more of BCL-XL, MCL, MDM2 and MDMX. The BCL-XL and MCLproteins are mitochondrial transmembrane proteins that prevent caspaseactivation by inhibiting the release of mitochondrial contents such ascytochrome c, leading to inhibition of apoptosis. MDM2 and/or MDMX,which are overexpressed in many cancer cells, are E3 ubiquitin-proteinligases, that bind to and promote degradation of p53. In some aspects,the fusion polypeptides of this invention mediate apoptosis by bindingto and disrupting and/or inhibiting the anti-apoptotic activity ofBCL-XL and MCL. In one aspect, the fusion polypeptides of this inventionalso mediate tumor-suppressive functions, including apoptosis, bydisrupting p53-interaction with MDM2 and/or MDMX, resulting inaccumulation of p53 in the cell.

The fusion polypeptides undergo HSA mediated transport into cells, and,in some aspects of this invention, inhibit the apoptosis inhibitorsBCL-XL and MCL, resulting in apoptosis regardless of p53 activity in thecell, e.g., p53-independent apoptosis activity. It has also surprisinglybeen found that the fusion polypeptides can mediate apoptosis andcytotixity in cells independent of the p53 genotype of the cell.Accordingly, in some aspects, the fusion polypeptides of this inventioncan mediate apoptosis and cytotixity in cells that are wild type forp53, as well as p53 mutant cells. p53 mutant cells include p53 negativecells, cells underexpressing p53. The fusion polypeptides of thisinvention also disrupt p53-MDM2 and/or p53-MDMX interactions, resultingin accumulation of cellular p53, which can then mediate apoptosis andinduce cytotoxicity. In some aspects, cells underexpressing p53 mayeither express low levels of p53, or express a p53 with partial orcomplete loss of function, i.e., lower or apoptosis activity compared towild-type p53, or may express low levels of p53. In some aspects, cellsmay overexpress p53, which may either express high levels of p53, orexpress a p53 with higher apoptosis activity than wild-type p53, orboth.

In one aspect, this invention relates to fusion polypeptides which arefirst in a class of compounds surprisingly demonstrated herein toinhibit two or more targets from two essential cellular pathwaysinvolved in modulating the tumor-suppressive functions, includingapoptosis. To the inventors' knowledge, no small molecule compounds haveyet been shown to efficiently inhibit two or more targets involved inapopotosis.

In one aspect the current invention provides a method of inhibitingBCL-XL and MCL-1 in a cell, the method comprising (a) providing a fusionpolypeptide or conjugate comprising a human serum albumin and ap53-peptide; and (b) contacting the cell with the fusion polypeptide,thereby inhibiting BCL-XL and MCL-1 inhibition of apoptosis. In someaspects inhibiting BCL-XL and MCL-1 disrupts BCL-XL and MCL-1 inhibitionof apoptosis. In some aspects of this invention, the cell contacted withthe fusion polypeptide is a cancer cell. It has been surprisinglydemonstrated that the fusion polypeptides of this invention can inhibitand/or disrupt, the inhibition of apoptosis mediated by BCL-XL andMCL-1, regardless of the p53 genotype of the cell, which can be a cancercell. In some aspects, the cell is a p53-wild type cancer cell. In someembodiments, the cell contacted with the fusion polypeptide or conjugateis a p53 mutant or a p53-negative cancer cell. In some aspects of thisinvention, the p53 mutant cancer cell contacted with the conjugate is acancer cell expressing low levels of p53, or a cell expressing a p53protein with lower BCL-XL/MCL-1 binding and/or lower apoptosis mediatingactivity than a wild-type p53 protein.

In some aspects of this invention, the fusion polypeptide used in themethod of inhibiting BCL-XL and MCL-1 in a cell and disrupting BCL-XLand MCL-1 inhibition of apoptosis is a recombinant fusion proteincomprising (a) a p53-derived peptide and a human serum albumin (HSA)polypeptide, or a fragment or variant of HSA; or (b) a p53-activatingpeptide and a human serum albumin In some embodiments, the fusionpolypeptide used in the method of inhibiting BCL-XL and MCL-1 in a cell,is a chemically cross-linked fusion polypeptide comprising (a) ap53-derived peptide and a human serum albumin; or (b) a p53-activatingpeptide and a human serum albumin The HSA polypeptide may comprise HSA,or a fragment or variant of HSA which retains its cell transport and/orligand binding properties, such as fatty acid binding. In some aspects,the HSA polypeptide may also comprise non peptide modifications. Theantibody polypeptide, e.g., antibody fragment polypeptide such as IG-Fcmay comprise an antibody, or a fragment or variant such as IG-Fc whichretains cell transport and/or ligand binding properties. In someaspects, the antibody polypeptide may also comprise non peptidemodifications. The transferrin polypeptide, e.g., may comprisetransferrin, or a fragment or variant which retains cell transportand/or ligand binding properties. In some aspects, the transferrinpolypeptide may also comprise non peptide modifications. In some aspectsof this invention, p53 agonists, including p53-peptides, for example,p53-derived peptides and p53-activating peptides, or theirpeptidomimetic or small molecule analogs may be useful for the methodsof this invention.

In some embodiments, the fusion polypeptide used in the method ofinhibiting BCL-XL and MCL-1 in a cell comprises one or more anticanceragents, in addition to the p53-derived peptide and a human serum albuminpolypeptide. In some embodiments, the anticancer agent is chemicallyconjugated to a natural ligand of human serum albumin In someembodiments, the additional anticancer agent is covalently bound to thea human serum albumin In some embodiments, the additional anticanceragent is bound to a human serum albumin polypeptide through non-covalentinteractions. In some embodiments, the anticancer agent is bound to afatty acid which is a natural ligand of human serum albumin In someembodiments, the additional anticancer agent is selected from the groupconsisting of 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, abiraterone acetate, afatinib, aldesleukin, alemtuzumab,alitretinoin, altretamine, amifostine, aminoglutethimide, anagrelide,anastrozole, anhydrovinblastine, arsenic trioxide, asparaginase,auristatin, azacitidine, azathioprine, bendamustine, bevacizumab,bexarotine, bicalutamide, bleomycin, BMS 184476, bortezomib, busulfan,cachectin, capecitabine, carboplatin, carmustine, cemadotin, cetuximab,chlorambucil, cisplatin, cladribine, crizotinib, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, dasatinib, daunorubicin,denileukin diftitox, decitabine,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide(SEQ ID NO: 5), docetaxel, dexamethasone, doxifluridine, doxorubicin,epirubicin, epoetin alpha, epothilone, erlotinib, estramustine,etinostat, etoposide, everolimus, exemestane, filgrastim, floxuridine,fludarabine, fluorouracil, fluoxymesterone, flutamide, folate linkedalkaloids, gefitinib, gemcitabine, gemtuzumab ozogamicin, GM-CT-01,goserelin, hexamethylmelamine, hydroxyureas, ibritumomab, idarubicin,ifosfamide, imatinib, interferon alpha, interferon beta, irinotecan,ixabepilone, lapatinib, leucovorin, leuprolide, lenalidomide, letrozole,lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, nelarabine, nilotinib,nilutamide, octreotide, ofatumumab, oprelvekin, oxaliplatin, paclitaxel,panitumumab, pemetrexed, pentostatin, polysaccharide galectininhibitors, procarbazine, raloxifene, retinoic acids, rituximab,romiplostim, sargramostim, sorafenib, streptozocin, sunitinib,tamoxifen, temsirolimus, temozolamide, teniposide, thalidomide,thioguanine, thiotepa, tioguanine, topotecan, toremifene, tositumomab,trametinib, trastuzumab, tretinoin, valrubicin, vegf inhibitors andtraps, vinblastine, vincristine, vindesine, vinorelbine, vintafolide,vorinostat, and a combination thereof.

In some aspects of this invention, inhibiting BCL-XL and MCL-1 in a cellusing the methods of this invention leads to death of the contactedcell. In some aspects of this invention, death of the cell contactedwith the fusion polypeptide occurs by apoptosis. It has beensurprisingly demonstrated that the fusion polypeptides of this inventioncan mediate apoptosis of the contacted cell, occurs independent of p53the genotype of the cell. For example, the cancer cell may be selectedfrom the group consisting of (a) a p53-positive cancer cell, (b) a p53mutant cancer cell, (c) p53-negative cancer cell, and (d) a cancer cellexpressing low levels of p53, or low activity p53. In some aspects, themethods further comprise disrupting p53-MDM2 and/or p53-MDMXinteractions. In some aspects, where the cells have a functional p53genotype/phenotype, disrupting p53-MDM2 and/or p53-MDMX leads toaccumulation of p53 in cells, which mediates apoptosis. In addition, thep53 peptide portion of the fusion polypeptide is also surprisingly shownto be capable of mediating apoptosis by inhibiting BCL-XL and MCL-1. Insome embodiments, cell death of the contacted cell occurs by apoptosis.

In another aspect the current invention provides a method of inducingcell death by disrupting BCL-XL or MCL-1 interactions with BAK and/or bydisrupting p53-MDM2 and/or p53-MDMX interaction, the method comprising(a) providing a fusion polypeptide comprising a human serum albuminpolypeptide, and a p53-peptide; and (b) contacting the cell with thefusion polypeptide. In some embodiments, the cell may be selected fromthe group consisting of (a) a p53-positive cancer cell, (b) a p53 mutantcancer cell, (c) p53-negative cancer cell, and (d) a cancer cellexpressing low levels of p53, or low activity p53. In some embodiments,the fusion polypeptide is (a) a recombinant fusion protein comprising ap53-derived peptide and a human serum albumin polypeptide, (b) arecombinant fusion protein comprising a p53-activating peptide and ahuman serum albumin polypeptide, (c) a chemically cross-linked fusionpolypeptide comprising a p53-derived peptide and human serum albumin, or(d) a chemically cross-linked fusion polypeptide comprising ap53-activating peptide and a human serum albumin polypeptide.

In another aspect, the current invention provides a method of treating asubject afflicted with a condition responsive to inhibiting BCL-XL, andMCL-1 and disrupting p53-MDM2 and/or p53-MDMX interaction, the methodcomprises (a) administration of therapeutically effective amount of afusion polypeptide comprising (i) a human serum albumin and ap53-peptide. The p53-peptide is a p53 derived peptide and/or ap53-activating peptide. In some embodiments, the subject is a human. Insome embodiments, the condition responsive to inhibiting BCL-XL, andMCL-1 and disrupting p53-MDM2 and/or p53-MDMX interaction is aneoplastic condition. In some embodiments, the condition responsive toinhibiting BCL-XL, and MCL-1 and disrupting p53-MDM2 and/or p53-MDMXinteraction is a cancer.

In some embodiments, the fusion polypeptide, used in the methods oftreating a condition responsive to inhibiting BCL-XL, and MCL-1 anddisrupting p53-MDM2 and/or p53-MDMX interaction, further comprises oneor more anticancer agents.

Thus, in some aspects, this invention also relates to: 1) making,screening and using p53-peptides optimized to inhibit BCL and MCL-1, andto disrupt p53-MDM2 and/or p53-MDMX interactions, in cancer therapy,regardless of the p53 genotype of the cancer cells; 2) developing smallmolecule compounds to mimic the function of p53-derived peptides andtarget p53 transcription dependent and independent pathwayssimultaneously; 3) combining p53-derived peptides or their analogueswith anticancer chemotherapeutics for synergistic efficacy. In vivomouse tumor model study showed that albumin-p53 activating peptidefusion protein can significantly boost the efficacy of MTX.

In some aspects, this invention relates to a pharmaceutical compositioncomprising a transporter protein and a p53 peptide such as a p53 derivedpeptide and/or a p53 activating peptide, optionally further comprising asmall molecule drug. In some embodiments, the transporter polypeptidemay be any natural or artificial polypeptide, including but not limitedto, for example, animal serum albumin, including but not limited to ahuman serum albumin (HSA) polypeptide, e.g., human serum albumin or afragment or variant thereof, animal serum globulin, including but notlimited to, an immunoglobulin, (an antibody) or antibody fragmentpolypeptide, such as an Ig-FC polypeptide, a transferrin polypeptide, anantennapedia peptide, cationic cell penetrating peptide (TAT),transportan and polyarginine. In some embodiments, this inventionrelates to a pharmaceutical composition for treating a neoplasticdisorder in an animal. In some embodiments, the neoplastic disorder is acancer. In some embodiments, the animal is a human. In some embodiments,the composition optionally includes one or more of pharmaceuticallyacceptable excipients, including but not limited to solvents, buffers,binders, disintegrants, fillers, glidants and lubricants. In someembodiments, the pharmaceutical composition is formulated as a capsule,tablet, pellet, dragee, semi-solid, powder, granule, suppositorie,ointment, cream, lotion, inhalant, injection, cataplasm, gel, tape, eyedrop, solution, syrup, aerosol, suspension, emulsion, or lyophilisate.

In some aspects, this invention relates to use of a fusion polypeptidecomprising a transporter protein and a p53 peptide such as a p53 derivedpeptide and/or a p53 activating peptide, and optionally a small moleculedrug, for the manufacture of a medicament for treating, alleviating orpreventing symptoms associated with a neoplastic disorder in an animal.In some embodiments, the transporter polypeptide may be any natural orartificial polypeptide, including but not limited to, for example,animal serum albumin, including but not limited to human serum albumin(HSA), or a fragment thereof, animal serum globulin, including but notlimited to, an immunoglobulin, (an antibody) or antibody fragmentpolypeptide, such as an Ig-FC polypeptide, a transferrin polypeptide, anantennapedia peptide, cationic cell penetrating peptide (TAT),transportan and polyarginine. In some embodiments, the neoplasticdisorder is a cancer. In some embodiments, the animal is a human. Insome embodiments, the medicament optionally includes one or more ofpharmaceutically acceptable excipients, including but not limited tosolvents, buffers, binders, disintegrants, fillers, glidants andlubricants. In some embodiments, the medicament is formulated as acapsule, tablet, pellet, dragee, semi-solid, powder, granule,suppositorie, ointment, cream, lotion, inhalant, injection, cataplasm,gel, tape, eye drop, solution, syrup, aerosol, suspension, emulsion, orlyophilisate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show design and purification of rHSA-P53i andrHSA-PMI. FIG. 1A: rHSA/peptide fusion protein was constructed by fusingeither P53i (SEQ ID NO: 1) or PMI (SEQ ID NO: 2) peptide sequence to theC-terminal of HSA. A caspase cleavage site (DEVDG (SEQ ID NO: 6)) wasincluded as a linker between HSA and peptide. FIG. 1B: Fusion proteinswere expressed using Pichia pastoris yeast expression system andpurified by cibacron blue dye agarose as described in Methods. Lane 1contains 10 μl of pre-stained Rec protein ladder (Fischer). Lanes 2 and3 contain 10 μg of purified rHSA-p53i and rHSA-PMI, respectively.Proteins were visualized by Coomassie blue staining, revealing >95%purity and MW of approximately 70 kD.

FIG. 2A, FIG. 2B, and FIG. 2C show that rHSA-p53 and rHSA-PMI areefficiently taken up into SJSA-1 cells. FITC-labeled rHSA (5 μM),FITC-rHSA-P53i (5 μM), and FITC-rHSA-PMI (5 μM) were added to SJSA-1cells as described in Methods. Visualization at 60× magnificationrevealed efficient uptake of FITC-rHSA (FIG. 2A), FITC-rHSA-P53i (FIG.2B) and FITC-rHSA-PMI (FIG. 2C) occurred following 24-hour incubation.FITC staining of vesicular cargo suggests significantly greater uptakeof rHSA-P53i and rHSA-PMI, compared to rHSA.

FIG. 3 shows that rHSA-P53i and rHSA-PMI bind to MDM2 and MDMX. Todetect the interaction between MDM2/MDMX and rHSA fusion proteins, 4 μgeach of biotinylated rHSA (lane 1), rHSA-P53i (lane 2), or rHSA-PMI(lane 3) were added to 200 μg of SJSA-1 whole cell lysate. MDM2 or MDMXantibody was added to the lysate followed by pulling down MDM2/MDMX andrHSA complexes using Protein A/G (1:1) resins. Samples were thenanalyzed by SDS-PAGE and Western blotting using MDM2, MDMX, andStreptavidin-HRP (Strep-HRP) antibodies.

FIG. 4A and FIG. 4B show that rHSA-P53i and rHSA-PMI promotecytotoxicity in SJSA-1 cells via caspase activation. rHSA fusionproteins, as well as nutlin (to serve as a p53-MDM2 antagonist control)were added at the indicated concentrations and allowed to incubate for24 hrs. FIG. 4A). Cytotoxicities were measured by CyQuant Assay andnormalized according to 10 μM rHSA-treated cells. FIG. 4B). Caspaseactivation was quantitated using the Homogeneous Caspase Assay asdescribed in Methods and normalized according to untreated cells.Results are displayed as percent cell death (FIG. 4A) or fold change(FIG. 4B) relative to 10 μM rHSA-treated wells. Data are representativeof 3 independent experiments performed in triplicate. Error barsindicate±SD.

FIG. 5 shows that rHSA-P53i and rHSA-PMI induce p53 accumulation, butnot MDM2. SJSA-1 cells were plated and allowed to attach overnight. Onday 2, culture media with 10 μM rHSA (lane 1), 10 μM nutlin (lane 2), 10μM rHSA-P53i (lane 3), or 10 μM rHSA-PMI (lane 4) were added torespective wells and allowed to incubate for 24 hrs. Cells were thenwashed, lysed and immunoblotted for p53 and MDM2. Western blot analysisto detect p53 protein (middle panel) reveals treatment with rHSA-P53i orrHSA-PMI resulted in modest accumulation of p53. Densitometry analysisreveals p53 accumulation following rHSA-P53i and rHSA-PMI treatment ison average, 1.5 and 2.9 orders of magnitude above control wells,respectively. As expected, nutlin-treatment promotes robust p53accumulation (11.5-fold average increase). However, unlike nutlin, whichpromotes a 5-fold increase in MDM2 expression, MDM2 protein remained atbasal levels following treatment with rHSA-P53i or rHSA-PMI (1.1- and1.0-fold change, respectively).

FIG. 6A and FIG. 6B show that rHSA fusion proteins are able to formstable complexes with FA-FITC. FIG. 6A). rHSA-PMI (lane 1-3), rHSA-P53i(lane 4-6) and rHSA (lane 7-9) were incubated at the indicated molarratios (rHSA:FA-FITC) with FA-FITC (lane 1, 4, and 7 (1:1); lane 2, 5,and 8 (1:2); lane 3, 6, and 9 (1:4); lane 10, FA-FITC only). The upperband in the gel corresponds to the HSA/FA-FITC complex, while the lowerband indicates unbound FA-FITC. Incorporation of FA-FITC into rHSA wasachieved up to a 1:4 rHSA:FA-FITC molar ratio. FIG. 6B). rHSA/FA-FITCcomplexes were pre-formed at a 1:4 molar ratio (HSA:FA-FITC; 30 pmol:120pmol) as described in Methods. Unlabeled FA was then added at theindicated concentrations to mimic the competition of free FA presentunder physiological conditions. The minimal dissociation of FA-FITC frompre-formed rHSA/FA-FITC complexes at the 8 times excess concentration ofunlabeled FA (lane 8) indicates FA-FITC and rHSA complex was highlystable in the presence of free FA. FIG. 6C). Preformed biotin-rHSA andFA-FITC complexes (biotin-rHSA:FA-FITC; 1:2) were incubated with PBS(Lane 1 and 3) and 10% serum (lane 2 and 4) for 1 and 24 hours. Lanes 1and 2 represent total FA-FITC incorporation into rHSA without and with10% FBS, respectively, prior to the addition of streptavidin resins.Lane 3 (with PBS only) and lane 4 (with 10% FBS) correspond to thesupernatants of samples after incubation with streptavidin resins andpulling down rHSA/FA-FITC complexes. The absence of rHSA/FA-FITCcomplexes in lane 3 indicates that all biotin-rHSA/FA-FITC complexes (inPBS) were efficiently pulled down. Any FA-FITC present in lane 4 wouldimply the displacement of rHSA-bound FA-FITC by serum components. Thepresence of only a weak band in lane 4 indicates the majority of FA-FITCremained bound to rHSA (pulled down by streptavidin resins).Quantitation of the amount of FA-FITC in lane 4 (Image J, NIH) revealedapproximately 15% and 17% of FA-FITC was removed from biotin-rHSA in thepresence of serum following 1 and 24 hour incubations, respectively.

FIG. 7A, FIG. 7B, and FIG. 7C show that rHSA/FA-FITC complexes retaininternalization and cytotoxic activity. FIG. 7A). FITC-labeled rHSA (5μM), FIG. 7B). rHSA/FA-FITC (5 μM/10 μM), and FIG. 7C). FA-FITC (10 μM)were added to SJSA-1 cells as described in Methods. Visualization at 60×magnification revealed efficient uptake of FITC-rHSA, rHSA/FA-FITC andFA-FITC occurred following 24-hour incubation. The extent of FITCstaining observed in rHSA/FA-FITC-treated cells is similar to that ofFA-FITC treatment alone indicating FA-FITC modification does not impairinternalization of rHSA. FIG. 7D). rHSA/FA-FITC or rHSA/FA-FITC fusionprotein complexes were added at a 1:2 molar ratio (rHSA:FA-FITC; 5μM:10μM) to SJSA-1 cells and allowed to incubate for 24 hrs. Nutlin (5 μM)was added in the presence of rHSA/FA-FITC to serve as a positivecontrol. Cytotoxicities were measured by CyQuant Assay as described inMethods. Results are displayed as percent cell death relative to 5 μMrHSA/FA-FITC-treated wells. Data are representative of an experimentperformed in triplicate. Error bars indicate±SD.

FIG. 8A and FIG. 8B show a schematic diagram of rHSA-mediated codeliverytechnology. Recombinant HSA-delivery complexes were conceived as aco-delivery technology in that 1) therapeutic peptides can be fused tothe C-terminal of HSA for both extracellular (FIG. 8A) and intracellular(FIG. 8B) targeting and 2) FA-Drugs can form stable complexes with rHSAfusion proteins to promote synergistic therapeutic efficacy.

FIG. 9A shows that that rHSA-P53i and rHSA-PMI bind to Bcl-xL and Mcl-1.To detect the interaction between Bcl-xL/Mcl-1 and rHSA fusion proteins,4 μg each of biotin-rHSA (lane 1), biotin-rHSA-P53i (lane 2), orbiotin-rHSA-PMI (lane 3) were added to 200 μg of MDA-MB-231 (a) SJSA-1(b) or Hela (c) whole cell lysates. Bcl-xL (FIG. 9A) or Mcl-1 (FIG. 9B)antibody was added to the lysate followed by pulling down Bcl-xL/Mcl-1and rHSA complexes using Protein A/G (1:1) resins. Samples were thenanalyzed by SDS-PAGE and Western blotting using Bcl-xL, Mcl-1, andStreptavidin-HRP (Strep-HRP) antibodies. Biotin-labeled HSA and HSA-p53iwere pulled down by streptavidin-conjugated agaroses (FIG. 9C). Proteinsassociated with HSA-p53i were blotted by BCL-XL and MCL-1 antibodies.

FIG. 10A, FIG. 10B, and FIG. 10C show that rHSA-P53i reduces Bak-Bcl-xLand BAK-MCL-1. rHSA-P53i reduces Bak-Bcl-xL (FIG. 10A-10C) and BAK-MCL-1(FIG. 10D) interactions in 3 cell lines. To determine whether rHSAfusion proteins were able to displace Bak from the BH3 binding groove ofBcl-xL, 4 μg each of biotin-rHSA (lane 1), biotin-rHSA-P53i (lane 2), orbiotin-rHSA-PMI (lane 3) was added to 200 μg of SJSA-1 (FIG. 10A) orHela (FIG. 10B) whole cell lysates. Bcl-xL or MCL-1 antibody was addedto the lysate followed by pulling down Bcl-xL or MCL-1 and rHSAcomplexes using Protein A/G (1:1) resins. Samples were then analyzed bySDS-PAGE and Western blotting using Bcl-xL, Bak, and Streptavidin-HRP(Strep-HRP) antibodies. Protein band quantitation was determined usingImage J software. HSA-p53i replaces BCL-XL or MCL-1-bound BAK. It isshown by decreased amount of BAK associated with BCL-XL or MCL.

FIG. 11 shows that rHSA-P53i attenuates Bak-Bcl-XL or MCL-1 interactionand promotes release of cytochrome c. HSA-p53i triggers apoptosis byreleasing cytochrome C from mitochondria to cytoplasm in cell lines.Cytosol cytochrome C is able to induce apoptosis.

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D show that rHSA-P53ico-localizes with mitochondria in SJSA-1 and Hela cells. FITC-labeledrHSA (5 μM) and FITC-rHSA-P53i (5 μM), were added to SJSA-1 and Helacells (green). Mitochondrial and nuclear staining was performed usingMitoTracker Deep Red (red) and Hoechst 33342 (blue), respectively.Visualization at 60× magnification revealed abundant yellow staining incells treated with rHSA-P53i (FIG. 12B and FIG. 12D), indicatingrHSA-P53i efficiently co-localized with mitochondrial organelles.Despite efficient rHSA uptake in all conditions, mitochondrialco-localization was not observed in cells treated with rHSA (FIG. 12Aand FIG. 12C).

FIG. 13A, FIG. 13B, and FIG. 13C show that Cytotoxic activity ofrHSA-P53i and rHSA-PMI is independent of p53 genotype. rHSA fusionproteins were added to MDA-MB-231 (FIG. 13A), SJSA-1 (FIG. 13B), or Hela(FIG. 13C) cells at the indicated concentrations and allowed to incubatefor 24 hrs. Nutlin (10 μM) served as a negative control in MDA-MB-231(p53-mutant) and Hela (unstable wild type p53) cells as it relies on awild type p53-dependent cytotoxic mechanism, and ABT-263 (ABT, 2.5 μM),a BH3 mimetic (Bcl-xL inhibitor), was included as a positive control toconfirm the presence of functional mitochondrial-mediated cytotoxicpathways. Cytotoxicities were measured by CyQuant Assay and normalizedaccording to 10 μM rHSA-treated cells. Results are displayed as percentcell death relative to 10 μM rHSA-treated wells. Data are representativeof 3 independent experiments performed in triplicate. Error barsindicate±SD.

FIG. 14A, FIG. 14B, and FIG. 14C show that HSA-PMI has synergisticeffect on SJSA-1 xenograft tumor model. HSA-PMI may induce apoptosisby 1) interrupting MDM2/MDMX and p53 interaction, 2) induce thereleasing BAK from MCL-1 or BCL-Xl and result in apoptosis. FIG. 14A andFIG. 14B show that co-administration of recombinant HSA-PMI and MTXenhances apoptosis compared to single agent administration in SJSA-1cells. FIG. 14C shows the synergistic efficacy of co-delivery rHSA-PMIand MTX.

FIG. 15 shows a schematic of exemplary p53 agonist-transporterpolypeptide compositions of this invention.

FIG. 16 shows a schematic of exemplary uses of the p53 agonists of thisinvention for the treatment of cancer and synergistic anti-cancereffects.

DETAILED DESCRIPTION

The current invention is based, in part, on our discovery that the p53agonists of this invention, for example, p53 peptides (not active p53),can bind and interact with several targets including, but not limitedto, MDM2, MDMX, BCL-XL, MCL p53 itself and MDM2, and induce cytotoxicityindependent on p53 genotype (wild type, mutation, or deletion).

In one aspect the current invention provides a method of inhibitingBAK-XL and MCL-1 in a cell, for example, by disrupting the BAK-BCL-XL orBAK-MCL-1 interaction in a cell, the method comprising (a) providing afusion polypeptide comprising a transporter polypeptide and ap53-agonist; and (b) contacting the cell with the fusion polypeptide. Inone aspect, the p53 agonist component of the fusion polypeptide is, insome aspects, a p53 peptide or small molecule agonist, for example, ap53 peptide, such as a p53 derived peptide or a p53 activating peptide,including peptide or peptidomimetic analogs thereof, or small moleculeanalogs thereof. The p53 agonists of this invention are capable ofinhibiting two or more targets from two essential cellular pathwaysinvolved in modulating apoptosis. In some aspects, the transporterpolypeptide is, for example, an HSA polypeptide, an antibody or antibodyfragment polypeptide, such as an Ig-FC polypeptide, a transferrinpolypeptide, an antennapedia peptide, cationic cell penetrating peptide(TAT), transportan and polyarginine. In one aspect the current inventionprovides a method of inhibiting BAK-XL and MCL-1 in a cell, for example,by disrupting the BAK-BCL-XL or BAK-MCL-1 interaction in a cell, themethod comprising (a) providing a fusion polypeptide comprising a humanserum albumin, and a p53-peptide; and (b) contacting the cell with thefusion polypeptide.

In one aspect, the instant disclosure provides a method of inhibitingBAK-XL and MCL-1 in a cell and inducing cell death in a cell, the methodcomprising (a) providing a fusion polypeptide comprising a transporterpolypeptide and a p53-agonist; and (b) contacting the cell with thefusion polypeptide. In one aspect, the p53 agonist component of thefusion polypeptide is, in some aspects, a p53 peptide or small moleculeagonist, for example, a p53 peptide, such as a p53 derived peptide or ap53 activating peptide, including peptide or peptidomimetic analogsthereof, or small molecule analogs thereof.

In one aspect, the instant disclosure provides a method of interactionbetween p53-MDM2 or p53-MDMX, the method comprising (a) providing afusion polypeptide comprising a transporter polypeptide and ap53-agonist; and (b) contacting the cell with the fusion polypeptide. Inone aspect, the p53 agonist component of the fusion polypeptide is, insome aspects, a p53 peptide or small molecule agonist, for example, ap53 peptide, such as a p53 derived peptide or a p53 activating peptide,including peptide or peptidomimetic analogs thereof, or small moleculeanalogs thereof.

In one aspect, the instant disclosure provides a method of interactionbetween p53-MDM2 or p53-MDMX and inducing cell death in a cell, themethod comprising (a) providing a fusion polypeptide comprising atransporter polypeptide and a p53-agonist; and (b) contacting the cellwith the fusion polypeptide. In one aspect, the p53 agonist component ofthe fusion polypeptide is, in some aspects, a p53 peptide or smallmolecule agonist, for example, a p53 peptide, such as a p53 derivedpeptide or a p53 activating peptide, including peptide or peptidomimeticanalogs thereof, or small molecule analogs thereof.

In one aspect, the instant disclosure provides a method of inhibitingBAK-XL and MCL-1 in a cell, and inhibiting the interaction betweenp53-MDM2 or p53-MDMX, the method comprising (a) providing a fusionpolypeptide comprising a transporter polypeptide and a p53-agonist; and(b) contacting the cell with the fusion polypeptide. In one aspect, thep53 agonist component of the fusion polypeptide is, in some aspects, ap53 peptide or small molecule agonist, for example, a p53 peptide, suchas a p53 derived peptide or a p53 activating peptide, including peptideor peptidomimetic analogs thereof, or small molecule analogs thereof.

In one aspect, the instant disclosure provides a method of inhibitingBAK-XL and MCL-1 in a cell, and inhibiting the interaction betweenp53-MDM2 or p53-MDMX and inducing cell death in a cell, the methodcomprising (a) providing a fusion polypeptide comprising a transporterpolypeptide and a p53-agonist; and (b) contacting the cell with thefusion polypeptide. In one aspect, the p53 agonist component of thefusion polypeptide is, in some aspects, a p53 peptide or small moleculeagonist, for example, a p53 peptide, such as a p53 derived peptide or ap53 activating peptide, including peptide or peptidomimetic analogsthereof, or small molecule analogs thereof.

In one aspect, the instant disclosure provides a method of inducing celldeath in a cell, the method comprising (a) providing a fusionpolypeptide comprising a transporter polypeptide and a p53-agonist; and(b) contacting the cell with the fusion polypeptide. In one aspect, thep53 agonist component of the fusion polypeptide is, in some aspects, ap53 peptide or small molecule agonist, for example, a p53 peptide, suchas a p53 derived peptide or a p53 activating peptide, including peptideor peptidomimetic analogs thereof, or small molecule analogs thereof.

In one aspect, the instant disclosure provides a method of treating asubject with cancer responsive to p53 inhibition of BCL-XL and MCL-1,and disrupting p-53 MDM2 interactions, the method comprising (a)providing a fusion polypeptide comprising a transporter polypeptide anda p53-agonist; and (b) contacting the cell with the fusion polypeptide.In one aspect, the p53 agonist component of the fusion polypeptide is,in some aspects, a p53 peptide or small molecule agonist, for example, ap53 peptide, such as a p53 derived peptide or a p53 activating peptide,including peptide or peptidomimetic analogs thereof, or small moleculeanalogs thereof.

In some embodiments, the instant disclosure provides a method oftreating a subject with cancer is selected from the group consisting ofAcute Lymphoblastic Leukemia (ALL), acute myeloid leukemia,adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,anal cancer, appendix cancer, astrocytoma, childhood cerebellar orcerebral cancer, basal-cell carcinoma, bile duct cancer, bladder cancer,bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstemglioma, brain cancer, brain tumor, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,Burkitt's lymphoma, carcinoid tumor, childhood tumor, carcinoid tumor,gastrointestinal, carcinoma of unknown primary, central nervous systemlymphoma, primary, childhood cerebellar astrocytoma, childhood cerebralastrocytoma/malignant glioma, cervical cancer, cholangiocarcinoma,chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, desmoplastic small round cell tumor, endometrialcancer, ependymoma, esophageal cancer, ewing's sarcoma in the ewingfamily of tumors, childhood extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, intraocular melanoma,eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach)cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor(GIST), germ cell tumor, extracranial germ cell tumor, extragonadal germcell tumor, ovarian germ cell tumor, gestational trophoblastic tumor,glioma of the brain stem, glioma, childhood cerebral astrocytoma,glioma, visual pathway and hypothalamic cancer, gastric carcinoidcancer, hairy cell leukemia, head and neck cancer, heart cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, non-Hodgkin lymphoma,hypopharyngeal cancer, hypothalamic and visual pathway glioma,intraocular melanoma, islet cell carcinoma (e.g. endocrine, pancreatic),Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer,leukaemia, acute lymphoblastic leukaemia (also called acute lymphocyticleukaemia), acute myeloid leukaemia (also called acute myelogenousleukemia), chronic lymphocytic leukaemia (also called chroniclymphocytic leukemia), chronic myelogenous leukemia (also called chronicmyeloid leukemia), hairy cell leukemia, lip and oral cavity cancer,liposarcoma, liver cancer (primary), lung cancer, non-small cell lungcancer, small cell lung cancer, AIDS-related lymphoma, Burkitt lymphoma,cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (anold classification of all lymphomas except Hodgkin's), primary centralnervous system cancer, macroglobulinemia, Waldenström, male breastcancer, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanoma, melanoma, intraocular (eye) cancer, merkelcell cancer, mesothelioma, adult malignant mesothelioma, metastaticsquamous neck cancer with occult primary, mouth cancer, multipleendocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm,mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, myelogenous leukemia,chronic, myeloid leukemia, adult acute, myeloid leukemia, childhoodacute myeloma, multiple myeloma, chronic myeloproliferative disorder,myxoma, nasal cavity and paranasal sinus cancer, nasopharyngealcarcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma,oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibroushistiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surfaceepithelial-stromal tumor), ovarian germ cell tumor, ovarian lowmalignant potential tumor, pancreatic cancer, pancreatic islet cellcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,pineal germinoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary adenoma, plasma cellneoplasia/multiple myeloma, pleuropulmonary blastoma, primary centralnervous system lymphoma, prostate cancer, rectal cancer, renal cellcarcinoma (kidney cancer), renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma, ewing family of tumors, sarcoma, kaposi, sarcoma, soft tissue,sarcoma, uterine, sézary syndrome, skin cancer (non-melanoma), skincancer (melanoma), skin carcinoma, merkel cell, small cell lung cancer,small intestine cancer, soft tissue sarcoma, squamous cell carcinoma,squamous neck cancer with occult primary, metastatic, stomach cancer,supratentorial primitive neuroectodermal tumor, T-cell lymphoma,cutaneous fungoides and sézary syndrome, testicular cancer, throatcancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,trophoblastic tumor, gestational, carcinoma of unknown primary site,cancer of unknown primary site, transitional cell cancer, urethralcancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer,visual pathway and hypothalamic glioma, childhood cancer, vulvar cancer,waldenström macroglobulinemia, and Wilms tumor (kidney cancer).

In some aspects, this disclosure relates to use of a fusion polypeptidedescribed herein, and optionally further small molecule drug, for themanufacture of a medicament for treating, alleviating or preventingsymptoms associated with a neoplastic disorder in an animal. In someembodiments, the transporter polypeptide may be any natural orartificial polypeptide, described above, including but not limited to,for example, animal serum albumin, polypeptide, including but notlimited to a human serum albumin (HSA) polypeptide, or a fragment orvariant thereof, animal serum globulin, including but not limited to, animmunoglobulin, (an antibody) or antibody fragment polypeptide, such asan Ig-FC polypeptide, a transferrin polypeptide, an antennapediapeptide, cationic cell penetrating peptide (TAT), transportan andpolyarginine. In some embodiments, the neoplastic disorder is a cancer.In some embodiments, the animal is a human. In some embodiments, themedicament optionally includes one or more of pharmaceuticallyacceptable excipients, including but not limited to solvents, buffers,binders, disintegrants, fillers, glidants and lubricants. In someembodiments, the medicament is formulated as a capsule, tablet, pellet,dragee, semi-solid, powder, granule, suppositorie, ointment, cream,lotion, inhalant, injection, cataplasm, gel, tape, eye drop, solution,syrup, aerosol, suspension, emulsion, or lyophilisate.

The fusion polypeptides of this invention have been shown to betransported into cells, and to surprisingly bind and interact with fourproteins involved in mediating apoptosis, from two essential cellularpathways involved in modulating apoptosis, including BCL-XL, MCL-1,MDM2, and MDMX. In some embodiments, the fusion polypeptides aretransported into the cell via HSA transport mechanisms.

In some embodiments, the fusion polypeptides of this inventionsurprisingly inhibit two or more targets from two essential cellularpathways involved in modulating apoptosis, for example, more than twotargets from two essential cellular pathways involved in modulatingapoptosis.

The p53 protein, a major cellular tumor suppressor, is situated at thecrossroads of a network of signaling pathways that are essential forcell growth regulation and apoptosis induced by genotoxic andnon-genotoxic stresses. In unstressed normal cells, the level of p53protein is regulated by binding of proteins such as MDM2 and MDMX thatpromote p53 degradation. After genotoxic stress, p53 proteinaccumulates, in part, because the inhibition of interaction of p53 withproteins such as MDM2 and MDMX and resulting downregulation ofdegradation of p53. p53 also gets activated and promotes it DNA repair,cell-cycle arrest, senescence, and apoptosis.

MDM2 and/or MDMX, which are overexpressed in many cancer cells. MDM2stands for mouse double minute 2 homolog, but the name “MDM2,” or “Mdm2”also encompasses other mammalian homologs, including human homolog. MDM2protein is an E3 ubiquitin-protein ligase that specifically binds theN-terminal trans-activation domain (TAD) of the p53 protein and therebypromotes ubiquitination and degradation of p53 protein. In addition,Mdm2 protein also functions as an inhibitor of p53 transcriptionalactivation. In some aspects, the fusion polypeptides of this inventionmediate the tumor-suppressive functions, including apoptosis bydisrupting p53-interaction with one or more of MDM2, MDMX and otherproteins that mediate p53 degradation, resulting in accumulation of p53in the cell. In some embodiments, the fusion polypeptides of thisinvention further inhibit the antiapoptotic activity of BAK-XL, MCL-1,or their homologs, orthologs or paralogs, for example, by disrupting theBAK-BCL-XL or BAK-MCL-1 interaction.

The BCL-XL, MCL and related proteins are mitochondrial transmembraneproteins that prevent caspase activation by inhibiting the release ofmitochondrial contents such as cytochrome c, leading to inhibitionapoptosis. BCL-2, BCL-XL and related proteins promote the survival ofneoplastic cell, in disorders including cancer and polycythemia vera.BCL-2 and BCL-XL may become overactive due to mutations in them or othergenes such as Jak2 mutations lead to over-activation of intracellularsignaling molecules, such as Stat5, which lead to transcription ofBcl-xL gene. In some aspects, the fusion polypeptides of this inventionmediate apoptosis by binding to and disrupting and/or inhibiting theanti-apoptotic activity of inhibiting BAK-XL, MCL-1, or their homologs,orthologs or paralogs in a cell, for example, by disrupting theBAK-BCL-XL or BAK-MCL-1 interaction.

In some aspects, p53 peptides of this invention are p53 derived peptidesor p53 activating peptides, including analogs thereof. In someembodiments, the invention provides fusion peptides comprising atransporter polypeptide and one or more p53 peptide, p53 activatingpeptides or small molecule agonists or inhibitors described herein. Insome embodiments, the p53 agonists of this invention are small moleculeanalogs of the p53 peptides, capable of inhibiting two or more targetsfrom two essential cellular pathways involved in modulating apoptosis.In some aspects, the fusion polypeptides inhibit BCL-XL and MCL and MDM2and/or MDMX, for example, the fusion polypeptides may inhibit one ormore of BCL-XL, MCL, MDM2 and MDMX. In some embodiments, the p53peptides or small molecule agonist of this invention are derived usingin vitro evolution for optimized binding to one or more of p53, BCL-XL,MCL, MDM2, MDMX, their orthologs, paralogs, homologs, analogs anddisrupting interaction between them, using or coupled with techniqueslike peptide display, phage display, mRNA display, ribosome display andthe like. In some embodiments, the p53 peptides or small moleculeagonists of this invention include peptides, peptide nucleic acids,nucleic acids and their analogs.

The p53 agonists, e.g., p53 peptides or p53 small molecule agonists,comprising the fusion polypeptides are capable of binding to two or moretargets from two essential cellular pathways involved in modulatingapoptosis. For example, in some embodiments, the p53 agonists, e.g., p53peptides or p53 small molecule agonists, comprising the fusionpolypeptides are capable of binding to BCL-XL, MCL, and/or to three ormore of BCL-XL, MCL-1, MDM2, and MDMX. For example, the p53 peptides insome embodiments, bind to BCL-XL and MCL-1, as well as to MDM2 and/orMDMX. In some embodiments, the “p53 peptide” may comprise a p53 peptide,for example, a peptide corresponding to amino acids 17-28 of the p53protein (ETFSDLWKLLPE, SEQ ID NO:1). In some embodiments, the “p53peptide” may comprise a p53 activating peptide, for example,TSFAEYWALLSP, SEQ ID NO:2.

In some embodiments, the p53-derived peptide is homologous or identicalto two or more contiguous amino acids of a region of p53 selected fromthe group consisting of activation domain 1 (amino acids 1-42), nuclearexclusion domain (amino acids 11-27), Highly Conserved Domain I (aminoacids 13-23), activation domain 2 (amino acids 43-92), proline richdomain (amino acids 64-92), DNA binding domain (amino acids 101-300),transcriptional activation domains (amino acids 1-42 or 55-75), MDM2binding domain, RPA binding domain, p62/TfB1 binding site (a subunit ofTFIIH), nuclear localization signaling domain (amino acids 316-325),homo-oligomerisation domain (amino acids 307-355) or the negativeregulatory domain (amino acids 356-393). In some embodiments, thep53-derived peptide may comprise one, two, three, four, five, six,seven, eight, nine or ten amino acid substitutions.

The p53 peptides of this invention are peptides are not full lengthactive p53. In some embodiments, the p53 peptide comprises a peptide oflength about 4 to about 100 amino acids homologous or identical tostretch of a vertebrate p53 peptide. For example, the p53-derivedpeptide may comprise, about 4-6, about 5-10, about 8-12, about 10-15,about 12-18, about 15-20, about 18-30, about 20-40, about 25-50, about30-60, about 40-80, about 50-100 amino acids. In some embodiments, thepeptides may comprise from 6 to 40 amino acids of p53, for example, fromabout 6 to about 35, from about 8 to about 35, from about 10 to about35, from about 10 to about 25, from about 10 to 20, about 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or any range of amino acids between anytwo of the recited numbers, from p53. In some embodiments, thep53-derived peptide is homologous or identical to one or more of human,mouse, rat, pig, cow, monkey, horse, cat, dog, or chicken p53, or p53from other domesticated, zoo, or aquatic mammals.

Examples of p53-derived peptide include but are not limited to FSDLWKLL(amino acids 19-26 of the p53 protein, SEQ ID NO:3), ETFSDLWKLLPE (aminoacids 17-28 of the p53 protein, SEQ ID NO:4).

The “p53 peptides” of this invention may also be peptidomimeticcompounds, or variants or analogs of peptides from regions of p53, or bemodified or derived from p53 peptides. In some embodiments, thep53-peptide may comprise non-naturally occurring amino acids. In someembodiments, the p53-peptide may comprise additional amino acids usefulfor one or more of spacer with another peptide, for altering stability,for altering solubility, for altering hydrophilicity/hydrophobicity, foraltering membrane permeability, for altering folding characteristics.

In some embodiments, the p53-peptide may be “derived” by an in vitroscreen for inhibition of a p53 function even if the p53 peptide is notdesigned based directly or entirely on a sequence from p53. The p53peptides comprising the fusion polypeptides may be capable of binding toBCL-XL, MCL, to three or more of BCL-XL, MCL, MDM2, and MDMX, or tothree or more proteins mediating apoptosis. In some embodiments, thep53-peptide may be derived from p53 itself. In some embodiments, the ap53-activating peptide may be derived from natural binding partners ofp53, including but not limited to p300/CBP, IKK alpha, S100B, p33ING1,14-3-3 zeta, p28ING4, 14-3-3 sigma, JNK1, JNK 2 JNK3, CDC2, MAPK, E4F1,PCAF, Cables, PKC alpha, ASPP2/53BP2, PKR, HSP90A, STE20 like kinaseMST1, HMG1, VRK1, Vimentin, Nucleolin, Rad51, AMF1, RecQ protein like 3,CCAAT-binding factor, RPA, DNA topoisomerase I, BRCA1, DNA topoisomeraseII alpha, BRCA2, HSF3, MDC1, Securin, Ref-1, PML, Pint, RNA polymeraseII EF, PTEN, Sin3A, ER alpha, Sp1, Mot-2, TAF9, Nucleostemin, WT1, HIF1alpha, ZBP89, WOX1, TRAP220, Ribonucleotide reductase, p53BP1, Mdm2,YB-1, MdmX, SMN1, ABL, p63, p73, ATM, SUMO1, CHK1, CHK 2, NEDD45, CK1alpha, E2-25K, CDK2, CDK5, CDK7, E2A, DNAPK, UBE3A, ERK1, ERK 2, HAUSP,GSK3 beta or Zinc-finger protein 363. In some embodiments, thep53-peptide may be derived from peptide library screening platform,including in vitro selection, using techniques but not limited to phagedisplay, ribosomal display, mRNA display, yeast display, against BCL-XL,MCL, MDM2, and MDMX.

The fusion polypeptides of this invention have been shown to betransported into cells, and to surprisingly bind and interact with morethan two target proteins involved in mediating apoptosis, includingBCL-XL, MCL, MDM2, and MDMX. In some embodiments, the transporterpeptides are conjugated with peptides capable of penetrating cells (cellpenetrating peptide). In some embodiments, the transporter peptides areconjugated with peptides capable of penetrating specific kind of cells,or a combination of a peptide capable of penetrating cell and anotherpeptide capable of recognition of a target cell, e.g., the cellsharboring one or more specific surface molecules or receptors.

In one aspect, this invention relates to fusion polypeptides which arefirst in a class of compounds surprisingly demonstrated herein toinhibit two or more targets from two essential cellular pathwaysinvolved in modulating the tumor-suppressive functions, includingapoptosis. To the inventors' knowledge, no small molecule compounds haveyet been shown to efficiently inhibit two or more targets involved inapopotosis.

In some embodiments, the p53 small molecule agonists of this inventionare synthesized, for example, using combinatorial chemistry methods, andscreened for binding to two or more targets from two essential cellularpathways involved in modulating apoptosis using high throughputscreening.

In some embodiments, the p53-peptide may be derived from p53. In someembodiments, the p53-peptide may bind one or more of the followingdomains of p53: activation domain 1 (amino acids 1-42), nuclearexclusion domain (amino acids 11-27), Highly Conserved Domain I (aminoacids 13-23), activation domain 2 (amino acids 43-92), proline richdomain (amino acids 64-92), DNA binding domain (amino acids 101-300),transcriptional activation domains (amino acids 1-42 or 55-75), MDM2binding domain, RPA binding domain, p62/TfB1 binding site (a subunit ofTFIIH), nuclear localization signaling domain (amino acids 316-325),homo-oligomerisation domain (amino acids 307-355) and the negativeregulatory domain (amino acids 356-393). In some embodiments, thep53-activating peptide has the sequence TSFAEYWNLLSP (SEQ ID NO: 7). Theamino acid sequence of human p53 is shown below:

Accession number/version number: AAD28535.1 GI:4731632 (SEQ ID NO: 8)  1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp 61 deaprmpeaa prvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe181 rcsdsdglap pqhlirvegn lrveylddrn tfrhsvvvpy eppevgsdct tihynymcns241 scmggmnrrp iltiitleds sgnllgrnsf evrvcacpgr drrtekenlr kkgephhelp301 pgstkralpn ntssspqpkk kpldgeyftl qirgrerfem frelnealel kdaqagkepg361 gsrahsshlk skkgqstsrh kklmfktegp dsd

In some embodiments, the p53-activating peptide may comprisenon-naturally occurring amino acids. In some embodiments, thep53-activating peptide may comprise additional amino acids useful forone or more of spacer with another peptide, which can, for example, beuseful for improving bioavailability, for example, for alteringstability, for altering solubility, for alteringhydrophilicity/hydrophobicity, for altering membrane permeability, foraltering folding characteristics.

In some embodiments, the fusion polypeptides undergo transport intocells, for example, HSA-mediated transport, and promote thetumor-suppressive functions, including apoptosis by disruptingp53-interaction with one or more of MDM2, MDMX and other proteins thatmediate p53 degradation, resulting in accumulation of p53 in the cell.In some aspects of this invention, apoptosis depends on havingfunctional p53 activity in the cell, including renaturation of p53.

In some embodiments, the fusion polypeptides undergo transport intocells, for example, HSA-mediated transport, and, in some aspects of thisinvention, inhibit the apoptosis inhibitors BCL-XL, MCL, or homologs,orthologs or paralogs thereof, resulting in apoptosis regardless of p53activity in the cell, e.g., p53-independent apoptosis activity. It hasalso surprisingly been found that the fusion polypeptides can mediateapoptosis and cytotoxity in cells independent of the p53 genotype of thecell. Accordingly, in some aspects, the fusion polypeptides of thisinvention can mediate apoptosis and cytotoxity in cells that are wildtype for p53, as well as p53 mutant cells. p53 mutant cells include p53negative cells, or cells underexpressing p53 or cells having lower leveror activity of p53. The fusion polypeptides of this invention alsodisrupt p53-MDM2 and/or p53-MDMX interactions, resulting in accumulationof cellular p53, which can then mediate apoptosis and inducecytotoxicity. In some aspects, cells underexpressing p53 may eitherexpress low levels of p53, or express a p53 with lower apoptosisactivity than wild-type p53, or both.

Therefore, in some embodiments, the fusion polypeptides of thisinvention induce cell death in cells including but not limited to (a) ap53-wild-type cell, (b) a p53 mutant cell, (c) a p53-negative cell,wherein the cell is a neoplatic cell, a cancerous cell, or aprecancerous cell.

In some embodiments, the cell contacted with the fusion polypeptide, isa cancer cell. In some embodiments, the cell is a p53-wild type cancercell. In some embodiments, the cell is a p53 mutant. In someembodiments, the p53 mutant cell is a p53-negative cancer cell withoutdetectable p53 activity, or a p53 mutant which expresses low levels ofp53, or a p53 with lower BCL, MCL-1, MDM2 and/or lower MDMX bindingactivity than wild-type p53.

In some embodiments, the present invention relates generally to fusionpolypeptides comprising a serum albumin polypeptide and a p53 peptide,and methods of treating, preventing, or ameliorating diseases ordisorders, such as cancer or other proliferative disorders. In someembodiments, the fusion polypeptide, used in method of disrupting theBak-Bcl-XL or BAK-MCL-1 interaction, is a recombinant fusion proteincomprising (a) one or more p53-peptides and a serum albumin polypeptide.The fusion polypeptides described herein comprise a polypeptide formedby the fusion of at least one molecule of albumin (or a fragment orvariant thereof) to at least one molecule of a p53-peptide. The p53peptide is a p53 derived peptide or a p53-activating peptide. Preferablythe serum albumin polypeptide is a human serum albumin polypeptide, or aserum albumin polypeptide derived from the species of the cells to becontacted or the subject to be treated. Reference to human serumalbumin, human serum albumin polypeptide, or rHSA also refers tofragments or variants thereof which maintain its cell transport functionand/or its ligand binding activity. Reference sequences for HSA and itsmature form are shown below.

1. Human Serum Albumin Preprotein

>gi|4502027|ref|NP_000468.1| serum albumin preproprotein [Homo sapiens](SEQ ID NO: 9) MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGEENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVADESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEPERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLKKYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAACLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAVARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADDRADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVENDEMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYARRHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVPDEFKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVPQVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVVLNQLCVLHEK TPVSDRVTKC CTESLVNRRP CFSALEVDETYVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHKPKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGL

2. Example of Mature Human Serum Albumin (Identical to aa 25-609 of theAbove)

>gi|332356380|gb|AEE60908.1| albumin, partial [Homo sapiens](SEQ ID NO: 10) DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHVKLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATLRETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEVDVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKRYKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKCASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTKVHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCEKPLLEKSHCI AEVENDEMRA DLPSLAADFV ESKDVCKNYAEAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKCCAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGEYKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKHPEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTESLVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKERQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGL

In one aspect, the current invention relates to fusion polypeptidescomprising a transporter polypeptide, and a p53 agonist, e.g., a p53peptide or small molecule agonist, for example, comprising a human serumalbumin (HSA) polypeptide and a p53 peptide. In some aspects, thetransporter polypeptide may be any natural or artificial polypeptide,including but not limited to, for example, animal serum albumin,polypeptide, including but not limited to a human serum albumin (HSA),or a fragment or variant thereof, animal serum globulin, including butnot limited to, an immunoglobulin, (an antibody) or antibody fragmentpolypeptide, such as an Ig-FC polypeptide, a transferrin polypeptide, anantennapedia peptide, cationic cell penetrating peptide (TAT, having thesequence YGRKKRRQRRR (SEQ ID NO: 11)), SynB1 (RGGRLSYSRRRFSTSTGR (SEQ IDNO: 12)), SynB3 (RRLSYSRRRF (SEQ ID NO: 13)), PTD-4 (PIRRRKKLRRLK (SEQID NO: 14)), PTD-5 (RRQRRTSKLMKR (SEQ ID NO:15)), FHV Coat-(35-49)(RRRRNRTRRNRRRVR (SEQ ID NO: 16)), BMV Gag-(7-25) (KMTRAQRRAAARRNRWTAR(SEQ ID NO: 17)), HTLV-II Rex-(4-16) (TRRQRTRRARRNR (SEQ ID NO: 18)),D-Tat (GRKKRRQRRRPPQ) (SEQ ID NO: 19), R9-Tat (GRRRRRRRRRPPQ (SEQ ID NO:20)), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 21)) chimera,MAP (KLALKLALKLALALKLA (SEQ ID NO: 22)), SBP(MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 23)), FBP(GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 24)), MPG(ac-GALFLGFLGAAGSTMGAWSQP KKKRKV-cya (SEQ ID NO: 25)), MPG^((ΔNLS))(ac-GALFLGFLGAAGSTMGAWSQPKSKRKV-cya (SEQ ID NO: 26)), Pep-1(ac-KETWWETWWTEWSQPKKKRKV-cya (SEQ ID NO: 27)), Pep-2(ac-KETWFETWFTEWSQPKKKR KV-cya (SEQ ID NO: 28)), polyarginines (R×N,where 2<N<27 (SEQ ID NO: 29)) chimera, and polylysines K×N (2<N<27 (SEQID NO: 30)) chimera. In some embodiments, the transporter peptide iscapable of binding to one or more ligands, wherein the ligand may be afatty acid, an amino acid, a nutrient, a vitamin, a metabolite, anhormone, a drug or a constituent of any bodily fluid. In someembodiments, the transporter peptide is a recombinant peptide. In someembodiments, the transporter peptide is conjugated with a chemicalentity, such as a peptide capable of traversing one or more of theplacental barrier, the blood-testis barrier, the blood-brain barrier, orany other blood-organ barrier that inhibits entry of the of thetransporter in any space within the body.

In some embodiments, the serum albumin polypeptide, for example the HSApolypeptide may also be truncated to modify the C-terminal region ofhuman serum albumin that binds to Fc receptor, in order to increaseretention of the fusion polypeptides and increase bioavailability. Thefusion proteins described herein are associated with one another, insome embodiments, recombinantly fused (e.g., an albumin open readingframe, encoding a human serum polypeptide comprising, for example, allor a portion of human serum albumin or variant thereof, is fused,in-frame, with a polynucleotide encoding a p53-peptide such as ap53-derived peptide or a p53 activating peptide). In some embodiments,p53-derived peptide or a p53-activating peptide are fused N-terminallyto a human serum albumin polypeptide. In some embodiments, p53-derivedpeptide or a p53-activating peptide are fused C-terminally to humanserum albumin In some embodiments, the p53-peptide may be fusedN-terminally and C-terminally to human serum albumin. In someembodiments, the p53-peptide may be inserted or fused in form of tandemrepeats of the p53-peptide. In some embodiments, other therapeuticpeptide(s) or protein(s) may be fused to human serum albumin p53-peptidefusion protein. In some embodiments, when p53 peptide is not fused toalbumin, it can be fused with other peptides including but not limitedto IgG Fc fragment, transferrin. In some embodiments, the p53-peptidemay be synthesized in tandem repeats and then conjugated to a serumalbumin or other suitable protein described herein. In some embodiments,p53-derived peptide or a p53-activating peptide are fused internally toa human serum albumin polypeptide. In some embodiments, multiple copiesof p53-derived peptide or a p53-activating peptide are fused to a humanserum albumin polypeptide. In some embodiments, only p53-derived peptideis fused with a human serum albumin polypeptide. In some embodiments,only p53-activating peptide is fused with a human serum albuminpolypeptide. In some embodiments, p53-derived peptide and p53-activatingpeptide is fused with a human serum albumin polypeptide. In someembodiments, a suitable linker of one to twenty-five amino acids areinserted at the junction of a human serum albumin polypeptide andp53-derived peptide or p53-activating peptide.

In some embodiments, the genetic fusion is cloned in a vector harboringof the invention may be expressed and purified from a suitable host suchas bacteria, yeast, insect cell lines, avian cell lines or mammaliancell lines.

In some embodiments, the fusion polypeptide, used in method ofdisrupting the Bak-Bcl-XL or BAK-MCL-1 interaction, is a chemicallycross-linked fusion polypeptide comprising a p53 peptide or agonist,fused to a human serum albumin polypeptide, for example, (a) ap53-derived peptide and human serum albumin, or (b) a p53-activatingpeptide and a human serum albumin polypeptide. In some embodiments,cross-linked fusion polypeptide comprising a human serum albuminpolypeptide and a p53-derived peptide or p53-activating peptide may beprepared using any suitable method known in the art. In someembodiments, Carbodiimide (e.g., EDC) may be used to chemicallycrosslink carboxyl groups to amine reactive groups to prepare across-linked fusion polypeptide comprising a human serum albuminpolypeptide and a p53-derived peptide or p53-activating peptide. In someembodiments, NHS ester, imidoester, pentafluorophenyl ester orhydroxymethyl phosphine may be used to chemically crosslinkamine-reactive groups to prepare a cross-linked fusion polypeptidecomprising a human serum albumin polypeptide and a p53-derived peptideor p53-activating peptide. In some embodiments, maleimide, haloacetyl(bromo- or iodo-), pyridyldisulfide, thiosulfonate, vinylsulfone may beused to chemically crosslink sulfhydryl-reactive groups to prepare across-linked fusion polypeptide comprising HSA or fragment or variantthereof and a p53-derived peptide or p53-activating peptide. In someembodiments, aldehyde-reactive groups like oxidized sugars (carbonyls)may be used to chemically crosslink hydrazide or alkoxyamine to preparecross-linked a comprising a human serum albumin polypeptide and ap53-derived peptide or p53-activating peptide. In some embodiments,diazirine, aryl azide n may be used to chemically crosslink for randominsertion to prepare a cross-linked fusion polypeptide comprising ahuman serum albumin polypeptide and a p53-derived peptide orp53-activating peptide. In some embodiments, isocyanate may be used tochemically crosslink hydroxyl (nonaqueous)-reactive groups to preparecross-linked fusion polypeptide comprising a human serum albuminpolypeptide and a p53-derived peptide or p53-activating peptide. In someembodiments, the p53 peptide, p53 agonist or fusion polypeptide may beconjugated to other chemicals, including but not limited to polyethyleneglycol, which in some embodiments may be conjugated to a human serumalbumin In some embodiments, the p53-peptide or the fusion polypeptideor conjugate described herein may be formulated in a particleformulation, for example, encapulated in liposomes, nanoparticles ormicroparticles. In some embodiments, the p53-derived peptide orp53-activating peptide or the fusion protein or conjugate may be boundto a nanoparticle or a microparticles. In some embodiments, the ratio ofp53-derived peptide or a p53-activating peptide to a human serum albuminpolypeptide may be anywhere in the range of about 100:1 to about 1:10.In some embodiments, only p53-derived peptide is fused with a humanserum albumin polypeptide. In some embodiments, only p53-activatingpeptide is fused with a human serum albumin polypeptide. In someembodiments, p53-derived peptide and p53-activating peptide is fusedwith a human serum albumin polypeptide. In some embodiments, a suitablelinker of one to twentyfive aminoacids are inserted at the junction of ahuman serum albumin polypeptide and p53-derived peptide orp53-activating peptide.

In some embodiments, the human serum albumin in the fusion polypeptidesdescribed herein may be substituted with serum albumin from othervertebrate species, including but to limited to, avian, bovine, canine,cervine, equine, ichthyic, feline, ovine, piscine and porcine albumin Insome embodiments the human serum albumin polypeptide in the fusionpolypeptides described herein may be substituted with other proteinsincluding but not limited to antibody polypeptides, such as IgG Fcfragment polypeptides, transferrin polypeptides, an antennapediapeptide, cationic cell penetrating peptide (TAT), transportan andpolyarginine

In some embodiments, the fusion polypeptide used in method of inhibitingor disrupting the Bcl-XL or MCL-1 inhibition of apoptosis comprises oneor more anticancer agents, in addition to the a p53-peptide and a humanserum albumin polypeptide. In some embodiments the anticancer agent ischemically conjugated to a natural ligand of human serum albumin In someembodiments, the additional anticancer agent is covalently bound to thea human serum albumin polypeptide. In some embodiments, the additionalanticancer drug is a small molecule drug. In some embodiments theadditional anticancer agent is bound to the a human serum albuminpolypeptide through non-covalent interactions. In some embodiments theadditional anticancer agent is bound to a fatty acid which is a naturalligand of human serum albumin In some embodiments, the additionalanticancer agent is selected from the group consisting of2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,abiraterone acetate, afatinib, aldesleukin, alemtuzumab, alitretinoin,altretamine, amifostine, aminoglutethimide, anagrelide, anastrozole,anhydrovinblastine, arsenic trioxide, asparaginase, auristatin,azacitidine, azathioprine, bendamustine, bevacizumab, bexarotine,bicalutamide, bleomycin, BMS 184476, bortezomib, busulfan, cachectin,capecitabine, carboplatin, carmustine, cemadotin, cetuximab,chlorambucil, cisplatin, cladribine, crizotinib, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, dasatinib, daunorubicin,denileukin diftitox, decitabine,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide(SEQ ID NO: 5), docetaxel, dexamethasone, doxifluridine, doxorubicin,epirubicin, epoetin alpha, epothilone, erlotinib, estramustine,etinostat, etoposide, everolimus, exemestane, filgrastim, floxuridine,fludarabine, fluorouracil, fluoxymesterone, flutamide, folate linkedalkaloids, gefitinib, gemcitabine, gemtuzumab ozogamicin, GM-CT-01,goserelin, hexamethylmelamine, hydroxyureas, ibritumomab, idarubicin,ifosfamide, imatinib, interferon alpha, interferon beta, irinotecan,ixabepilone, lapatinib, leucovorin, leuprolide, lenalidomide, letrozole,lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, nelarabine, nilotinib,nilutamide, octreotide, ofatumumab, oprelvekin, oxaliplatin, paclitaxel,panitumumab, pemetrexed, pentostatin, polysaccharide galectininhibitors, procarbazine, raloxifene, retinoic acids, rituximab,romiplostim, sargramostim, sorafenib, streptozocin, sunitinib,tamoxifen, temsirolimus, temozolamide, teniposide, thalidomide,thioguanine, thiotepa, tioguanine, topotecan, toremifene, tositumomab,trametinib, trastuzumab, tretinoin, valrubicin, vegf inhibitors andtraps, vinblastine, vincristine, vindesine, vinorelbine, vintafolide,vorinostat, and a combination thereof. Combinations of the fusionpolypeptide of this invention with any one or more of the anticanceragents recited above is featured in this invention.

In some embodiments, inhibiting Bcl-XL or MCL-1, leads to death of thecell by apoptosis. In some embodiments, the apoptosis in the cell occursindependent of p53 genotype (wild-type or mutant).

In another aspect the current invention provides a method of inducingcell death in a cell by disrupting or inhibiting BCL-XL, MCL-1 anddisrupting p53-MDM2 and/or p53-MDMX interactions, the method comprises(a) providing a fusion polypeptide comprising a a human serum albuminpolypeptide and a p53-peptide; and (b) contacting the cell with thefusion polypeptide, thereby inducing cell death. In some embodimentscell death occurs through apoptosis In some embodiments, the cell is acancer cell selected from the group consisting of (a) a p53-wild-typecancer cell; or (b) a p53 mutant cancer cell, such as a p53-negativecancer cell or a cancer cell expressing low levels of p53 or a p53having lower BCL-XL, MCL-1 or MDM2/MDMX binding activity than p53.

In some embodiments, the fusion polypeptide used in the method ofinducing cell death by disrupting or inhibiting BCL-XL, MCL-1 anddisrupting p53-MDM2 and/or p53-MDMX interactions is (a) a recombinantfusion polypeptide comprising a p53-derived peptide and a human serumalbumin polypeptide; (b) a recombinant fusion polypeptide comprising ap53-activating peptide and a human serum albumin polypeptide; (c) achemically cross-linked fusion polypeptide comprising a p53-derivedpeptide and a human serum albumin polypeptide; or (d) a chemicallycross-linked fusion polypeptide comprising a p53-activating peptide andHSA or fragment or variant thereof.

In another aspect, the current invention provides a method of treating asubject with a condition responsive to disrupting or inhibiting BCL-XL,MCL-1 and disrupting p53-MDM2 and/or p53-MDMX interactions, the methodcomprising (a) administering a therapeutically effective amount of afusion polypeptide comprising (i) a human serum albumin polypeptide anda p53-derived peptide, or (ii) a human serum albumin polypeptide and ap53-activating peptide. In some embodiments, the subject is a human. Insome embodiments, the condition responsive to disrupting or inhibitingBCL-XL, MCL-1 and disrupting p53-MDM2 and/or p53-MDMX interactions, is acancer. In some embodiments, the fusion polypeptide, used in the methodsherein further comprises one or more therapeutic agent, such as ananti-cancer agent.

In some aspects, this invention relates to a pharmaceutical compositioncomprising a transporter protein and a p53 peptide such as a p53 derivedpeptide and/or a p53 activating peptide, optionally further comprising asmall molecule drug. In some embodiments, the transporter polypeptidemay be any natural or artificial polypeptide, including but not limitedto, for example, animal serum albumin polypeptide, polypeptide,including but not limited to a human serum albumin (HSA), or a fragmentor variant thereof, animal serum globulin, including but not limited to,an immunoglobulin (an antibody) or antibody fragment polypeptide, suchas an Ig-FC polypeptide, a transferrin polypeptide, an antennapediapeptide, cationic cell penetrating peptide (TAT), transportan andpolyarginine. In some embodiments, this invention relates to apharmaceutical composition for treating a neoplastic disorder in ananimal. In some embodiments, the neoplastic disorder is a cancer. Insome embodiments, the animal is a human. In some embodiments, thecomposition optionally includes one or more of pharmaceuticallyacceptable excipients, including but not limited to solvents, buffers,binders, disintegrants, fillers, glidants and lubricants. In someembodiments, the pharmaceutical composition is formulated as a capsule,tablet, pellet, dragee, semi-solid, powder, granule, suppositorie,ointment, cream, lotion, inhalant, injection, cataplasm, gel, tape, eyedrop, solution, syrup, aerosol, suspension, emulsion, or lyophilisate.

In some embodiments, the compositions of current invention comprise offusion polypeptides described herein, wherein the fusion peptide issubstantially purified. In some embodiments, the substantially fusionpeptides of the invention are lyophilized and formulated with a bufferor a preservative.

Pharmaceutical Compositions, Doses, and Administration

In one embodiment the fusion polypeptide comprising a human serumalbumin polypeptide and a p53 peptide (for example, a p53-derivedpeptide or p53-activating peptide) may be formulated in form ofpharmaceutical composition for administering to a subject in needthereof. The compositions suitable for use in the method of currentinvention may be formulated in using one or more physiologicallyacceptable carriers or excipients. The compositions may be formulated assolutions in appropriate solvents suitable for use in the method ofcurrent inventions. In one embodiment the fusion polypeptides comprisinga human serum albumin polypeptide and p53-derived peptide orp53-activating peptide may be formulated in form of an aqueous solutionprepared using a carrier such as physiologically acceptable osmogen orbuffer solution as including but not limited to saline, phosphatebuffered saline and water. In one embodiment the non-saline osmogen mayadvantageously be an amino acid selected from a group comprisinghistidine, valine, proline and cysteine. The osmogen may also be: apolyalchohol phosphoric ester such as glycerophosphate, a polyol such asmannitol, sorbitol, glycerol or xylitol, a monosaccharide such asglucose, galactose, xylose, fructose, galactosamine, glucosamine,neuraminic acid, and glucuronic acid; or a disaccharide such as sucrose,maltose and lactose. In one embodiment the fusion polypeptidescomprising a human serum albumin polypeptide and p53-derived peptide orp53-activating peptide may be formulated as a non-aqueous solventsolution for pharmacologic use. In some embodiments, the non-aqueoussolvent solution can be prepared by dissolving the fusion polypeptide ina solvent comprising DMSO, or lipid carrier.

The formulations of this invention comprising the fusion polypeptidecomprising a human serum albumin polypeptide and p53-derived peptide orp53-activating peptide may also comprise excipients and/or carriersselected from solubilizers, stabilizers, buffers, tonicity modifiers,bulking agents, viscosity enhancers/reducers, surfactants, chelatingagents, and adjuvants.

The pharmaceutical compositions of this invention may comprise thefusion polypeptides present at a concentration in the range of about0.000001 to about 10% (weight/volume), for example, from about 0.000001to about 0.0001%, from about 0.0001% to about 0.01%, from about 0.01% toabout 0.1%, from about 0.1% to about 1%, from about 1% to about 10%(weight/volume).

In one embodiment, the fusion polypeptide comprising a human serumalbumin polypeptide and p53-derived peptide or p53-activating peptidemay be formulated for parenteral delivery by a route such asintravenous, subcutaneous, intramuscular, and intra-articularadministration. These formulations are either liquids or lyophilizates.In one embodiment, the formulation comprising the fusion polypeptide isprepared as a concentrate, which is diluted with a suitable carrierbefore administration. In one embodiment, the formulation comprising thefusion polypeptide is prepared as a lyophilised powder, which is dilutedwith a suitable carrier before administration. The liquid or lyophilizedformulations may comprise from 1-50% of the fusion polypeptidescomprising a human serum albumin polypeptide and p53-derived peptide orp53-activating peptide. The lyophilized formulations of this inventionmay also comprise excipients and/or carriers selected from solubilizers,stabilizers, buffers, tonicity modifiers, bulking agents, viscosityenhancers/reducers, surfactants, chelating agents, and adjuvants.Lyophilized formulations need to be reconstituted prior toadministration. These ingredients are well known to one of ordinaryskill in the art. Liquid formulations are optionally diluted withpharmaceutically acceptable diluents such as 5% Dextrose Injection, USPor 0.9% Sodium Chloride Injection, USP. These formulations areadministered by infusion or bolus administration.

In one embodiment, the formulations of this invention comprising thefusion polypeptide comprising a human serum albumin polypeptide and ap53-peptide, such as a p53 derived peptide or p53-activating peptide canalso be formulated for oral delivery as a solution, gelatin capsule, ortablet. The oral liquid formulations and capsule formulations are wellknown to one of ordinary skill in the art. The tablet formulation caninclude: 1-80% the fusion polypeptides comprising a human serum albuminpolypeptide and a p53-derived peptide or p53-activating peptide; 10-90%binders, disintegrants, fillers, glidants, lubricants; and 1-20%additional compounds that ensure easy disintegration, disaggregation,and dissolution of the tablet in the stomach or the intestine.

The tablet may be formulated for immediate release, sustained release,or delayed or modified release. The tablet may be optionally coated canmake the tablet resistant to the stomach acids and it disintegrates inthe duodenum, jejunum and colon as a result of enzyme action or alkalinepH. These formulations are well known to one of ordinary skill in theart. The tablets may be further coated with sugar, varnish, or wax tomask the taste.

In certain embodiments, pharmaceutical compositions comprisingformulation comprising the fusion polypeptides comprising a human serumalbumin polypeptide and a p53-derived peptide or p53-activating peptidemay be formulated for administration by other routes of administration,including but not limited to systemic peripheral, or topicaladministration. Illustrative routes of administration include, but arenot limited to, oral, transdermal, transmucosal, intranasal, ocular,pulmonary, rectal, vaginal, parenteral, such as by injection, includingsubcutaneous, intradermal, intramuscular, intravenous, intraarterial,intracardiac, intrathecal, intraspinal, intracapsular, subcapsular,intraorbital, intraperitoneal, intratracheal, subcuticular,intraarticular, subarachnoid, and by implant of a depot or reservoir,such as intramuscularly. Dosage of the pharmaceutical compositions mayvary by route of administration. Certain administration methods mayinclude the step of administering the composition one or more times aday to obtain the desired therapeutic effect.

In one embodiment, the formulation may comprise the fusion polypeptidescomprising a human serum albumin polypeptide and p53-derived peptide orp53-activating peptide is prepared as an aerosol balm, cream, emulsion,foam, gel, liniment, lotion, ointment, suspension or spray.

In one embodiment, the fusion polypeptides comprising a human serumalbumin polypeptide and a p53-derived peptide or p53-activating peptideare formulated as a topical composition, which may includepharmaceutically acceptable excipients. Exemplary excipients may besolvents (e.g. water, alcohol, propylene glycol, ethylene glycol,glycerol), hydrocarbon bases (e.g., hard paraffin, soft paraffin,microcrystalline wax and ceresine), absorption bases (e.g., wool fat,beeswax), water soluble bases (e.g., macrogols 200, 300, 400),emulsifying bases (e.g., emulsifying wax, cetrimide), emu oil, vegetableoils (e.g., olive oil, coconut oil, sesame oil, almond oil and peanutoil), polymers (e.g. dextran, polyacrylic acid, carbomer, polyethyleneoxide, polyethylene glycol, a copolymer of ethylene oxide and propyleneoxide, polyvinylpyrrolidone, arabinogalactan), cellulose (e.g.hydroxypropylmethyl cellulose, carboxypropylmethyl cellulose,methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose,hydroxypropylmethyl cellulose), gums (e.g., guar or xanthan gum), salts,acids, bases etc., or penetrant (e.g., dimethyl sulfoxide, dimethylacetamide, dimethylformamide and n-decyl-methyl sulfoxide propyleneglycol, glycerin, lanolins, alcohols, anionic emulsifiers (e.g. sodiumlauryl sulfate) and surfactants (nonionic emulsifiers such aspolyoxyethylene fatty alcohol ethers and esters; polyoxyethylene fattyacid esters, e.g. polyoxyethylene stearate; polyoxyethylene sorbitanfatty acid esters and sorbitan fatty acid esters, e.g. sorbitanmonostearate; polyoxyethylene glycol fatty acid esters; polyol fattyacid esters, e.g. glyceryl monostearate; and ethoxylated lanolinderivatives).

METHODS OF THE INVENTION

In one aspect, the current invention provides method of treating asubject with a condition responsive to inhibiting or disrupting BCL-XLand MCL-1 mediated inhibition of apoptosis, and disrupting p53-MDM2and/or p53-MDMX interaction, comprising administering a compositioncomprising a fusion polypeptide of this invention. The methods includeadministering, to a patient, a composition (e.g., a pharmaceuticalcomposition) comprising a fusion polypeptide comprising a human serumalbumin polypeptide and a p53-peptide, such as a p53-derived peptide orp53-activating peptide. Administration by one or more of the followingroutes: oral, epidural, intraarticular, intracardiac, intracavernous,intradermal, intramuscular, intraosseous, intraperitoneal, intrathecal,intravenous, intravitreal, nasal inhalation, oral, subcutaneous,topical, and is contemplated.

The invention also features the use of fusion polypeptides comprising ahuman serum albumin polypeptide and a p53-derived peptide orp53-activating peptide in the preparation of a medicament for use in anyof the methods described herein. Also included are the uses of fusionpolypeptides comprising a human serum albumin polypeptide and ap53-derived peptide or p53-activating peptide in the preparation of amedicament for the treatment of a condition responsive to inhibiting ordisrupting BCL-XL and MCL-1 mediated inhibition of apoptosis, anddisrupting p53-MDM2 and/or p53-MDMX interaction. Also included are theuses of fusion polypeptides comprising a human serum albumin polypeptideand a p53-derived peptide or p53-activating peptide in the preparationof a medicament for the treatment of cancer.

Use of proteins and peptides as therapeutic agents has investigated inrecent years, with the average number of new peptide drug candidatesgrowing from an average of 1.2 per year in the 1970's to 16.8 per yearso far in the 2000's [1]. These biologically active molecules haveadvantages over small molecule drugs, including higher specificity anddecreased potential to cause adverse effects. Among these promisingcandidates, however, few are known to bind intracellular proteins, thusignoring potentially clinically relevant intracellular targets. Anefficient cell penetrating technology remains one of the major obstaclesto peptide drug administration. In some embodiments, this inventionfeatures a delivery technology using a human serum albumin polypeptide,for example, recombinant human serum albumin (rHSA), to promote cellularpenetration of a therapeutic peptide, optionally together with a smallmolecule drug. In addition to prolonging serum stability, this novelstrategy is capable of facilitating simultaneous intracellular deliveryof two therapeutic agents, each with distinct but complimentarymechanisms, to promote a synergistic therapeutic response for thetreatment of a variety of diseases. One model used to test this deliverytechnology employed a p53-derived peptide to target the anti-apoptoticinteraction between two intracellular proteins, p53 and MDM2. The p53tumor suppressor protein plays a critical role in generating cellularresponses to a number of stress signals, including DNA damage, aberrantproliferative signals due to oncogene activation, and hypoxia. Uponactivation, p53 is stabilized and moves to the nucleus, where it bindsto DNA in a sequence specific manner and promotes transcriptionalregulation of genes involved in DNA repair, cell-cycle arrest,senescence, and apoptosis [2,3]. Previous studies demonstrated thatp53-mediated apoptosis plays a critical role to suppress tumor formationin mice [4].

While it is estimated that the p53 gene is mutated in 50% of tumors,increasing evidence reveals that a large percentage of tumors retainwild type p53, but possess other alterations in the p53 pathway, whichprevents its critical tumor-suppressive function [5]. One key componentaltering p53 activity is the E3 ubiquitin ligase, MDM2. This negativeregulator directly binds to p53 and promotes the ubiquitination andsubsequent proteasomal degradation of p53. Under normal conditions, MDM2functions as a harness for p53 activity, regulating its subcellularlocation, transcriptional activity, and stability. In tumors, however,MDM2 is frequently upregulated, thus preventing the p53 stress responseeven in cases where wild type p53 is present. As a result, patientsoften display accelerated tumor growth and a diminished response totreatment [6,7]. Disruption of the p53-MDM2 interaction has become apopular strategy to increase functional p53 levels and thus, reducecancer cell viability.

The binding interface of p53-MDM2 is composed of a hydrophobic cleft onthe N-terminal surface of MDM2 and the N-terminal transactivation domainof p53. Since the revealing of the interaction interface, a series ofsmall molecules and peptides have been developed to target thep53-binding pocket of MDM2 [8]. One such class of small moleculeantagonists termed nutlins, have demonstrated the ability to dock withinthe p53-binding pocket of MDM2, resulting in p53 accumulation,initiation of cell cycle arrest, and ultimately, apoptosis [9,10].Despite this, translation into an effective treatment modality has shownlittle promise. The limited effect of small molecule p53-MDM2 inhibitorsis thought to be in part due to the lack of inhibition of MDMX, ahomolog of MDM2 [11,12]. In depth analyses of MDM2 and MDMX revealedboth proteins work in concert to decimate the p53 pathway, thusnecessitating the development of an inhibitor with dual specificity[13]. Rationally designed synthetic peptides offered an alternative tonutlins and other small molecule antagonists by binding and inhibitingboth MDM2 and MDMX. PMI peptide was developed by Li and colleagues tocompete with p53 for MDM2 and MDMX binding at an affinity approximately2 orders of magnitude higher than that of a wild type p53-derivedpeptide (containing amino acids 17-28 of the p53 protein) [14,15]. Asrecognized herein, although this work provided support for targeting thep53-MDM2/MDMX interaction as a cancer therapy, problems surroundingproteolytic stability and intracellular peptide delivery still remained.

Interest in using HSA as a drug carrier has grown in recent years due toa number of properties including: preferential uptake in tumor andinflamed tissue, stability, biodegradability, ready availability, andlack of toxicity and immunogenicity [16-18]. HSA is capable of improvingthe pharmacokinetic profile of peptide- or protein-based drugs bychemical conjugation, genetic fusion, and micro/nano particleencapsulation [19]. HSA is the most abundant plasma protein with anaverage half-life of 19 days. It functions as a natural transportvehicle for metal ions, a number of drugs, and long chain fatty acids inthe blood [20]. In addition, tumor cells often have an increased rate ofalbumin uptake. For example, HSA makes up 19% of the soluble proteinswithin certain breast cancer cells [21]. A number of drugs have beendesigned to exploit these valuable characteristics of HSA. Acylatedinsulin and glucagon-like peptide-1, which rely on HSA-mediated bindingto extend serum stability, have been approved for clinical use [22,23].The HSA/paclitaxel nanoparticle known as Abraxane was approved for thetreatment of metastatic breast cancer and has shown promise as adelivery strategy to extend the half-life and therapeutic efficacy ofsmall molecule drugs [19]. A major concern of albumin-based formulationssuch as Abraxane, however, is the uncertainty surrounding their aptitudefor generating an immune response against endogenous HSA. Based onextensive in vivo studies and several clinical trials, no such immuneresponse has been reported, even for HSA fused to immunostimulatingcytokines, such as interferon a2b (Albuferon) [24]. Furthermore,Recombumin, a genetically engineered form of HSA used to replaceendogenous albumin, is already in therapeutic use and has a proven lackof toxicity and immunogenicity [17].

The cell penetration technology described herein utilizes the long-chainfatty acid transport properties of HSA with the methods herein forgenetically modifying HSA to deliver a highly specific peptide to anintracellular target. Acylation with long chain FA is a method that haspreviously been used to extend the serum half-life of small compounds byfacilitating nonspecific association with serum albumin and lipoproteins[25]. In contrast to the acylated drugs currently approved for clinicaluses that rely on random serum protein association in vivo, ourHSA-mediated delivery technology is pre-formulated under optimized invitro conditions to guarantee simultaneous intracellular delivery of twocomplimentary therapeutic agents. This strategy allows drug transportand release to mimic the robust fatty acid uptake as well as albumintransport.

Combination therapy is one approach for cancer treatment. Delivery ofmultiple therapeutics, simultaneously to one target, can improveefficacy and minimize toxicity. Encapsulated micro/nano particles andconjugated polymers have been developed to co-deliver differenttherapeutics. However, as discussed herein, those methods are plagued bya number of factors including immunogenicity, difficulty in penetratingsolid tumors, lack of selectivity for target tumor tissue, inefficientdissociation from a covalently-bound carrier, and reliance on passivediffusion, a process that does not guarantee co-delivery of bothanticancer agents to the same cell [26,27]. The data described hereinsuggests HSA is a feasible choice to serve as a protein carrier forco-delivery of C-terminal fused p-53 peptides and FA-modified molecules.In the realm of cancer treatment, it is our hope that such a system canultimately be used to facilitate intracellular delivery of twoanticancer agents, each with distinct roles in regard to triggering orresponding to cellular DNA damage, to promote a more robust apoptoticresponse for the treatment of solid tumors.

As discussed herein, the results demonstrate the feasibility of usinggenetically modified HSA to fuse a therapeutic p53-peptide, whileretaining FA-binding ability for use as a carrier to co-deliver both ap53-peptide and FA-modified Drug (FA-Drug). Two exemplary fusionpolypeptides, e.g., HSA proteins containing either a wild typep53-derived peptide (P53i) (SEQ ID NO:1) or the high affinityMDM2-binding peptide N8A-PMI (PMI) (SEQ ID NO:2) were cloned, expressedin Pichia pastoris yeast system, and purified [28]. Cellular andbiochemical studies indicate that rHSA-P53i and rHSA-PMI wereefficiently taken up by osteosarcoma SJSA-1 cells and retained MDM2- andMDMX-binding activity. In addition, both rHSA-P53i and rHSA-PMI promotedcytotoxicity in SJSA-1 cells via caspase activation. As the futureapplication of this rHSA delivery technology aims to deliver one or moreFA-Drugs in addition to a C-terminal-fused therapeutic peptide,FA-binding and stability studies were also performed using FA-FITC. Ashypothesized by the inventors, exemplary fusion polypeptides (e.g., rHSAproteins (rHSA-P53i and rHSA-PMI)) were able to form highly stablecomplexes with FA-FITC via non-covalent interactions. In addition,FA-FITC complexed with HSA could be internalized by the target cells andrHSA fusion proteins still retained cytotoxicity.

EXAMPLES Example 1 Methods for Design and Expression of Exemplary FusionPolypeptides, rHSA-p53 and rHSA-PMI

Recombinant HSA fusion proteins were cloned into pPICZαA vector(Invitrogen). The 5′ primer contained a 21 base pair sequenceoverlapping with the N-terminal of HSA cDNA and the XhoI cloning site ofthe vector (5′-ATCGCTCGAGAAAAGAGAGGCTAAGCGACGCACACAAGAGTGAGGTTGCT-3′(SEQ ID NO: 31)). The 3′ primer contained a portion of the C-terminal ofHSA, wild type P53i (ETFSDLWKLLPE (SEQ ID NO: 1)) or PMI (TSFAEYWALLSP(SEQ ID NO: 2)) peptides, as well as the NheI restriction site. Thissequence was subsequently amplified by PCR. Each peptide sequence wasfused to the C-terminal of HSA by overlapping PCR with primers:

(SEQ ID NO: 32) 5′- CCATAGGTCTGAAAACGTTTCACCTCAACTTCGTCGGCGCCTAAGGCAGCTTGACTTGCAGC-3′ (HSA C-terminal), (SEQ ID NO: 33) 5′-CGATGCTAGCACTAGTTTATTCAGGAAGTAGTTTCCATAGGTCTGAAAAC GTTTCACC-3′(rHSA-P53i, C-terminal), (SEQ ID NO: 34) 5′-CGATGCTAGCCCGCGGTTATGGACTAAGAAGAGCCCAGTACTCAGCAAAACTTGTACCGTCAACTTCGTCGGCGCC-3′ (rHSA-PMI, C- terminal).

The pPICZαA vector was digested with XhoI and XbaI to create the 5′ and3′ cloning sites. The HSA fusion protein sequences were digested withXhoI and NheI and ligated into the linearized vector. Following ligationand transformation, the cloned genes were confirmed by DNA sequencing.Pichia pastoris yeast cells (Invitrogen, 18258-012) were thentransformed using linearized pMM1 (for HSA-P53i) or pMM2 (for rHSA-PMI)plasmid DNA and rHSA-P53i and rHSA-PMI clones were selected for Zeocinresistance after 72 hours. Recombinant proteins were then expressed inPichia pastoris according to the manufacturer's instructions(Invitrogen, K1740-01).

Purification of rHSA-P53i and rHSA-PMI

Culture media containing albumins secreted from P. pastoris werefiltered (0.2 μm) and subsequently incubated for 4 hours at 4° C. withcibacron blue dye agarose (Sigma, C9534) [29]. Following 10× volumewashes with ice-cold PBS, recombinant proteins were eluted stepwiseusing PBS containing sodium thiocyanate (NaSCN) (100 mM, 200 mM, 500 mM,750 mM and 1 M). Fractions containing rHSA-P53i or rHSA-PMI (500 mM to750 mM NaSCN) were pooled and dialyzed in PBS. Purified proteins wereanalyzed by SDS-PAGE with purity greater than 95%. Recombinant HSAproteins were then filtered (0.2 μm) for sterilization and stored at−20° C. Protein concentration was determined using the Bradford method(Bio-Rad, 23225).

FITC and Biotin-Labeling of rHSA

FITC and biotin-labeling of rHSA (biotin-rHSA) were performed usingNHS-Fluorescein (Thermo Scientific, 46409) and NHS-Biotin (ThermoScientific, 20217) according to the manufacturer's instructions. Bothlabeling procedures were performed using 20 times excess of NHS.Following the incubation, proteins were dialyzed in 1× PBS to removeunconjugated NHS reagents.

Cell Culture and Cytotoxicity Studies

Osteosarcoma SJSA-1 cells (ATCC) were grown in RPMI media containing 10%FBS. Cytotoxicity assays were performed using SJSA-1 cells plated in 24-or 96-well plates at 20,000 or 5,000 cells per well, respectively. Cellswere allowed to attach overnight. All treatments were added on day 2 inRPMI media containing 1% FBS plus the equivalent amounts of 1× PBSbuffer. Unless otherwise indicated, cells were exposed to treatmentmedia for 24 hours, at which time cytotoxicity was measured using thefluorometric CyQuant assay (Invitrogen, C35006) or the fluorometricHomogeneous Caspase assay (Roche, 03005372001), according to themanufacturer's instructions, for detection of apoptosis. All resultswere plotted relative to rHSA- or where indicated, rHSA/FA-FITC-treatedcells.

Confocal Microscopy

On day 1, SJSA-1 cells were seeded in 6-well plates, at a density of80,000 cells per well, and allowed to attach overnight. Treatment mediacontaining 1% FBS plus 5 μM FITC-labeled rHSA (FITC-rHSA),FITC-rHSA-P53i or FITC-rHSA-PMI was added to wells on day 2 and allowedto incubate for 24 hours. For experiments to examine the internalizationactivity of FA-FITC-modified rHSA, SJSA-1 cells were plated as describedabove. On day 2, rHSA fusion proteins (dissolved in 1× PBS) wereincubated with FA-FITC at a 1:2 molar ratio (rHSA:FA-FITC; 5 μM:10 μM)to allow formation of rHSA/FA-FITC complexes. Reactions were conductedin PBS at room temperature for 30 minutes, prior to dilution in RPMImedia (without FBS) and addition to wells. Following a 24 hourincubation period, cells were trypsinized, re-plated onto coverslips andallowed to re-attach for 2 hours. Upon re-attachment, cells were washed3× with PBS and maintained in phenol red-free media. For visualization,imaging was performed using a Nikon TiE (Eclipse) confocal microscopewith a CSU-X spinning disk confocal scan head (Yokogawa), a linearencoded x, y robotic stage (ASI Technologies, Inc.), equipped with amulti-bandpass dichromatic mirror and bandpass filters (ChromaTechnology Corp.) in an electronic filter wheel for selection of FITC.488 nm laser illumination was provided by a 50 mW monolithic lasercombiner (MLC400, Agilent Technologies) and images were acquired using a60×1.40NA objective and the Clara interline CCD camera (AndorTechnology).

Co-Immunoprecipitation and Protein Detection by Western Blotting

SJSA-1 cells were lysed in buffer containing 20 mM Tris-HCl, 50 mM NaCl,0.05% Triton X-100, and protease inhibitor cocktail. For each condition,200 μg SJSA-1 cell extract was heated to 42° C. prior to the addition of4 μg biotinylated rHSA-P53i, rHSA-PMI or rHSA protein. Mixtures werethen allowed to incubate for 1 hour at RT. Next, MDM2 (Santa Cruz,sc-965) or MDMX antibody (Santa Cruz, sc-74467) was added to each tubeand allowed to incubate, while rotating, for 4 hours at 4° C. Proteinsbound to MDM2/MDMX antibody were pulled down using Protein A/G (1:1)resins and samples were analyzed by SDS-PAGE and Western blotting usingMDM2, MDMX, and Streptavidin-HRP antibodies (Pierce, 21130). Primary andsecondary antibodies were added in 2% non-fat dry milk in TBST at thefollowing dilution ratios: p53 (Santa Cruz, sc-126), MDM2, MDMX (1:200),streptavidin-HRP (1:2500), and GAPDH (1:5000) (Santa Cruz, sc-59541).Proteins were visualized by the chemiluminescent detection solution,SuperSignal West Dura Extended Duration Substrate (Thermoscientific,34075) and densitometry was performed on replicate experiments usingImage J software (NIH).

Gel Shift Assays to Determine Stability of FA/HSA Complexes

All recombinant albumins were defatted following a previous publication[30]. FA-FITC was synthesized by mixing 1× 1-Hexadecylamine and 2×N,N-Diisopropylethylamine (Sigma) followed by addition of 1×NHS-Fluorescein (Thermo Scientific). This reaction was carried outovernight, protected from light. Products were then purified by HPLC andidentified by MS. For experiments designed to detect FA/HSA complexformation, rHSA (30 pmol, dissolved in 1× PBS) was incubated with orwithout FA-FITC at desired molar ratios and then separated by 0.5× TBSPAGE. Due to the incorporation of fluorescent molecules, HSA bound byFA-FITC in gel can be visualized under UV. Assays to determine thedegree of displacement of FA-FITC were performed by adding an excessamount of unlabeled FA to pre-formed rHSA/FA-FITC complexes, at theindicated molar ratios. Reactions were conducted in PBS containing 10%glycerol at room temperature for 30 minutes. The products were separatedusing non-denaturing 0.5× TBS PAGE and then visualized under 305 nm UV.Experiments to determine the effect of the presence of serum on HSA/FAcomplexes were conducted using pre-formed biotin-rHSA/FA-FITC complexes.After initial complex formation, the biotin-rHSA/FA-FITC solution wasdivided equally among 4 tubes. Ten percent FBS or the same volume of PBSwas subsequently added to respective samples and the mixtures wereallowed to incubate for up to 24 hours at 37° C. followed by theaddition of streptavidin-conjugated resins to pull-down biotinylatedrHSA/FA-FITC complexes (GenScript, L00353). Streptavidin resins werepre-equilibrated with HSA to minimize nonspecific interactions. Samplesperformed in parallel to assess total FA-FITC incorporation did notreceive streptavidin resins, but underwent an identical incubation timeof either 1 or 24 hours at 37° C. Next, samples were centrifuged to pulldown biotin-HSA/FA-FITC and a portion of each supernatant was analyzedusing 0.5× TBS PAGE as above.

Example 2 HSA Fusion Protein Design, Expression, and Purification

The structural basis for MDM2/MDMX interaction with p53 is discussedbelow. To form a complex with MDM2 or MDMX, the amphipathic a-helixfragment of the N-terminal transactivation domain of p53 must bindwithin the concave binding pocket of MDM2/MDMX. Despite minor sequencedifferences and a slightly smaller hydrophobic binding cleft in MDMXcompared to MDM2, structural studies reveal that the p53 binding domainsof both proteins display a high degree of similarity. The minimallyrequired MDM2/MDMX binding sequence includes residues 19-26 of thetransactivation domain of wild type p53 (F19S20D21L22W23K24L25L26 (SEQID NO: 3)). Three critical residues, known as the hydrophobic triad(F19W23L26), bind to the three distinct sites of the MDM2/MDMXhydrophobic pocket [8,31,32].

In this study, two peptide sequences were chosen as exemplary sequencesto test the initial HSA-mediated peptide delivery approach. The firstconstruct fused to HSA contains the wild type p53 binding sequence(E17T18F19S20D21L22W23K24L25L26P27E28 (SEQ ID NO: 1)) and the secondsequence is a potent MDM2/MDMX peptide inhibitor, PMI (TSFAEYWNLLSP (SEQID NO: 7)), adopted from the work of Li and colleagues [14,15]. To avoidthe effect of bulky HSA structure on peptide-MDM2/MDMX interaction, acaspase cleavage site (DEVDG (SEQ ID NO: 6)) was inserted as a linkerbetween HSA and peptide (FIG. 1A). The insertion of this linker mayfacilitate liberation of peptides from HSA following p53 accumulationand subsequent caspase activation.

The wild type p53-derived peptide (P53i) or PMI peptide sequences werefused to the C-terminal of HSA (FIG. 1A) using a protocol as describedin Methods. The fusion proteins were then cloned into pPICZαA Pichiapastoris protein expression vectors and transformed into yeast cells.Recombinant HSA-P53i and rHSA-PMI were overexpressed and purified usingcibacron blue dye agarose to achieve >95% purity confirmed by SDS-PAGE(FIG. 1B).

Example 3 Both rHSA-P53i and rHSA-PMI are Efficiently Internalized bySJSA-1 Cells

Therapeutic activity hinges upon successful delivery of a peptide orsmall molecule drug into the cell. While new strategies are consistentlybeing evaluated to deliver functional proteins or peptides into cells,they are still lacking in overall efficiency and safety for translationinto a clinical model [1]. Current HSA drug formulations such asAbraxane, the HSA-paclitaxel nanoparticle, demonstrate efficientintracellular HSA uptake. Multiple modes of internalization have beenshown to play a role, including receptor-mediated as well as endocyticpathways [19].

While the exact mechanism underlying the uptake of rHSA-P53i andrHSA-PMI is not fully known, the results provided herein confirm thatexemplary fusion polypeptides, such as rHSA fusion proteins, are in facttaken up by cells, a critical step to target intracellular proteins. Todo this, confocal microscopy was employed to visualize the extent ofinternalization of FITC-labeled rHSA fusion proteins by SJSA-1 cells.Cells were treated with 5 μM FITC-rHSA, FITC-rHSA-P53i or FITC-rHSA-PMIin the presence of 1% FBS for 24 hours. Depicted in FIG. 2A-C, all threeproteins (rHSA, rHSA-P53i, and rHSA-PMI) were taken up into SJSA-1cells. In comparison to rHSA, treatment with rHSA-P53i and rHSA-PMIresulted in robust intracellular vesicle formation and distribution.

Example 4 Exemplary Fusion Polypeptides, rHSA-P53i and rHSA-PMI, BindBoth MDM2 and MDMX

Exemplary fusion polypeptides, rHSA-P53i and rHSA-PMI, were designed toelicit inhibitory activity against MDM2 and its homolog, MDMX [8, 9, 10,32], in order to disrupt p53-MDM2, and result in accumulation of p53 andrestoration of its tumor-suppressive function. To confirm both exemplaryfusion polypeptides possess MDM2/MDMX binding ability, SJSA-1 whole celllysate was incubated in the presence of biotin-rHSA, biotin-rHSA-P53i orbiotin-rHSA-PMI. Proteins were then pulled down using anti-MDM2 oranti-MDMX antibodies and followed by Western blotting usingstreptavidin-HRP to detect biotinylated rHSA bound to MDM2/MDMX.Reciprocal detection was performed using streptavidin resins to pulldown biotinylated rHSA protein followed by Western blotting usinganti-MDM2 or anti-MDMX antibodies (data not shown). As depicted in FIG.3, both rHSA-P53i and rHSA-PMI co-immunoprecipitated with MDM2 and MDMX,thus confirming that target protein binding ability was retainedfollowing peptide fusion to HSA, and transport into the cell.

Example 5 Exemplary Fusion Polypeptides, rHSA-P53i and rHSA-PMI, PromoteCytotoxicity via Caspase Activation

Adequate levels of p53 are necessary to mediate the cytotoxic effectsfrom chemotherapy or radiation treatment and restoration of its activityhas been found to promote tumor regression in mice [4,33]. A smallmolecule p53-MDM2 antagonists, nutlins, has previously been used to showthat competition with endogenous p53 at the hydrophobic binding cavityof MDM2, can result in accumulation of p53 and initiation of apoptosis[9,10]. To determine if exemplary polypeptides, such as the rHSA fusionproteins, are able to trigger apoptosis via p53 activation, theMDM2-overexpressing cell line, SJSA-1, was incubated with rHSA-P53i,rHSA-PMI or nutlin. Following a 24-hour treatment, cytotoxicity wasassessed using the CyQuant Assay. Both rHSA-P53i and rHSA-PMI promotedcytotoxic responses in SJSA-1 cells (approximately 60% and 84% celldeath, respectively) (FIG. 4A).

p53 functions as a tumor suppressor by promoting the expression ofpro-apoptotic proteins capable of triggering apoptosis via caspaseactivation. To determine if the cytotoxic response observed was indeedoccurring as a result of an apoptotic mechanism, SJSA-1 cells weretreated as described above and analyzed for caspase activation. Resultsin FIG. 4B reveal an approximate 7-fold increase in caspase activationin rHSA-PMI-treated cells and up to 2-fold increase in all othertreatments. These data confirm that both exemplary fusion polypeptides,rHSA-P53i and rHSA-PMI, promoted cytotoxicity in SJSA-1 cells and thisresponse was driven by apoptotic mechanisms.

Example 6 Exemplary Fusion Polypeptides, rHSA-P53i and rHSA-PMI, Promotep53 Accumulation

p53 functions as a transcription factor for genes involved in mediatingkey cellular processes such as, DNA repair, cell-cycle arrest,senescence, and apoptosis. In addition, p53 upregulates MDM2 proteinexpression, via an autoregulatory feedback loop [34-36]. The results inFIG. 3 illustrate that rHSA-P53i and rHSA-PMI are capable of disruptingnative p53-MDM2 interaction. To extend these studies, the effect ofexemplary fusion polypeptides, such as the rHSA fusion proteins, on p53and MDM2 protein expression was determined. In FIG. 5A, Western blottingwas performed to detect both MDM2 and p53 protein levels in SJSA-1 cellsafter incubation with rHSA-P53i, rHSA-PMI, nutlin, or media alone for 24hours. As expected, both rHSA-P53i and rHSA-PMI promoted a mild increasein p53, 1.5- and 2.9-fold increases, respectively. Nutlin-treatmentpromoted robust p53 accumulation (11.5-fold average increase). However,while nutlin displayed a 5-fold average increase in MDM2 proteinexpression relative to untreated cells; the exemplary fusionpolypeptides, rHSA-P53i and rHSA-PMI, did not promote any significanteffects on MDM2, resulting in only 1.1- and 1.0-fold changes in proteinlevels, respectively. Thus, it was surprisingly found that maintenanceof lower MDM2 levels following administration of exemplary fusionpolypeptides, such as rHSA fusion protein treatment, may confer anadvantage over nutlin, as upregulation of MDM2 may counterbalanceincreases in p53 protein and thus, compromise therapeutic efficacy [37].

Example 7 Fatty Acid (FA)-Modified FITC Forms a Stable Complex withExemplary Fusion Polypeptides, rHSA-P53i and rHSA-PMI

FA modification has previously been used to prolong the half-life ofsmall compounds by facilitating non-specific association with serumalbumin and lipoproteins [25]. Here, the potential of using rHSA fusionproteins to deliver a FA-Drug was examined Unlike acylated drugscurrently in use [22,23], the inventors used an in vitro formulationstrategy, whereby a FA-Drug is incorporated into rHSA prior toadministration. This method ensures uniform and reproducible complexformation, and guarantees each rHSA protein administered will co-deliverboth FA-Drugs and a therapeutic peptide.

In this study, the extent of incorporation and stability of pre-loadedFA using FA-FITC was examined Recombinant HSA/FA-FITC complex formationwas detected using a gel shift assay as described in Methods. Completeincorporation of FA-FITC was achieved up to a 1:4 ratio of rHSA:FA-FITC.Notably, the degree of FA binding was similar among rHSA, rHSA-P53i, andrHSA-PMI (FIG. 6A). This implies rHSA fusion proteins folded properlyand the C-terminal peptide fusion did not alter FA binding ability. Asnative albumin and free FA will be present under physiologicalconditions, we designed experiments to mimic an in vivo setting in orderto assess the overall stability of rHSA/FA complexes. These FAcompetition assays included: 1) determination of the extent of exchangeof HSA-bound FA with excess free FA and 2) assessment as to whether ornot incubation in the presence of serum would displace FA frompre-formed rHSA/FA complexes. Our data indicate that rHSA/FA-FITCcomplexes (formed at 1:4 molar ratio; rHSA:FA-FITC) were stable in thepresence of unlabeled FA, up to a 1:8 molar ratio (rHSA-associatedFA-FITC:unlabeled FA) (FIG. 6B). Next, we examined the extent ofexchange of rHSA-bound FA-FITC with lipoproteins and albumin present inserum. To do this, pre-formed biotinylated rHSA/FA-FITC complexes weredivided into four separate reaction mixtures. Each reaction conditioncorresponds to the lane assignments (lanes 1-4), as depicted in FIG. 6C.Samples 2 and 4 received 10% serum, while samples 1 and 3 received thesame volume of PBS. Each mixture was then allowed to incubate for up to24 hours at 37° C. To determine the degree of dissociation of FA-FITCfrom pre-formed rHSA/FA-FITC complexes into serum proteins,streptavidin-conjugated resins were added to samples 3 and 4. Thisallowed biotin-rHSA/FA-FITC to be pulled down and the supernatants wereanalyzed for the presence of FA-FITC by gel shift assay. The samealiquots were taken from samples 1 and 2 to serve as positive controlsfor the determination of total HSA-bound FA-FITC present in each sampleprior to the addition of streptavidin-conjugated resins. Any appearanceof FA-FITC in lanes 3 and 4 represented the amount of FA-FITC thatdissociated from pre-formed rHSA/FA-FITC complexes. A comparison betweenlane 1 (total rHSA-bound FA-FITC) and lane 4 (serum-associated FA-FITC)indicates the degree of FA-FITC displacement from rHSA into serumproteins. Approximately 86% of FA-FITC remained bound to rHSA followinga 24-hour incubation, demonstrating only minimal exchange of FA-FITCoccurred between pre-formed rHSA/FA-FITC and albumin or lipoproteins inserum.

Example 8 FA-FITC and Exemplary Fusion Polypeptide Complexes (Such asrHSA Fusion Protein Complexes) were Able to Transport FA-FITC andPromote Cytotoxicity

Acylated drugs currently approved for clinical use rely on HSA toenhance solubility and mediate transport to locations within thevicinity of target tissue. Studies have shown that while no grossstructural disorganization occurs upon FA incorporation into HSA, FAshave been observed to stabilize the protein against denaturation duringclinical applications, indicating some subtle structural changes mayoccur [38]. It has been demonstrated that FA-FITC and HSA form a stablecomplex. However, it is uncertain whether the complex interferes withthe cellular uptake of FA-FITC or the cytotoxic activity of rHSA fusionproteins.

Confocal microscopy was employed to examine internalization ofrHSA/FA-FITC complexes as described in Methods. Prior to imaging, SJSA-1cells were incubated with FITC-labeled rHSA, pre-formed rHSA/FA-FITCcomplexes, or FA-FITC alone (FIG. 7A-C, respectively). As illustrated inFIG. 6, we have determined that rHSA/FA-FITC complexes are highly stableeven in the presence of 10% serum. The results in FIG. 7 demonstrateuptake of FA-FITC, pre-formulated with rHSA (FIG. 7B), is similar tothat of FA-FITC directly added to the culture medium (FIG. 7C). Thediffuse FITC staining in FIG. 7B indicates HSA/FA-FITC complexes do notinterfere with uptake of FA-FITC or affect intracellular distribution ofFA-FITC. To assess the effect of FA-FITC on rHSA fusion proteins,rHSA/FA-FITC, rHSA-P53i/FA-FITC, and rHSA-PMI/FA-FITC complexes wereformed at a molar ratio of 1:2 (rHSA:FA-FITC; 5 μM:10 μM). Thesecomplexes, as well as a positive control containing nutlin plusrHSA/FA-FITC, were added to SJSA-1 cells and allowed to incubate for 24hours. The results in FIG. 7D reveal the cytotoxic effects of both ofthe exemplary fusion polypeptides, rHSA-P53i and rHSA-PMI, complexedwith FA-FITC. The cytotoxicity associated with rHSA-P53i/FA-FITC andrHSA-PMI/FA-FITC was comparable to that of 5μM rHSA-P53i and rHSA-PMI(FIG. 4A).

Example 9 Discussion

The advent of small peptide therapeutic agents has resulted in theability to enhance target specificity and blunt toxicity compared tosmall molecule drugs. Despite these advantages, serum instability andrapid renal clearance have plagued their widespread usage. This exampledemonstrates the feasibility of using a therapeutic fusion polypeptideas an efficient peptide delivery method to target intracellularproteins, circumvent proteolytic degradation in vivo, and translate intoenhanced serum stability and improved therapeutic efficacy. In addition,this delivery technology has been designed to exploit the intrinsicfatty acid transport properties of HSA to allow it to co-deliver both aFA-Drug and an intracellular-targeting p53 peptide (such as a p53derived peptide or a p53 activating peptide, e.g. P53i or PMI) to elicita synergistic therapeutic response (FIG. 8).

HSA possesses three homologous domains. Based on physicochemicalstudies, HSA is a highly flexible protein that is capable of changingits molecular shape under different conditions. The flexibility ispartially due to the relative motions of its domain structures. Inparticular, the two alpha helices in the C-terminal of domain III haveminimal interaction with other parts of the protein [16]. The C-terminalis thus the logical location for sequence fusion of therapeuticpeptides. The data presented here, using a p53 reactivation model,support our hypothesis that rHSA can be genetically engineered todeliver a therapeutic peptide to an intracellular target and serve as acarrier for FA-modified small molecule drugs. Wild type p53 (P53i) andPMI peptides were fused into HSA by genetic cloning, expressed in aPichia pastoris yeast system, and purified (FIG. 1). Intracellularuptake of rHSA-P53i and rHSA-PMI by MDM2-overexpressing SJSA-1 cells wasconfirmed by confocal microscopy (FIG. 2A-C). Furthermore,co-immunoprecipitation assays revealed rHSA fusion proteins were capableof occupying the hydrophobic binding pocket of both MDM2 and MDMX, thuspreventing native p53 degradation (FIG. 3). These actions resulted inthe accumulation of p53 and subsequently, apoptosis (FIGS. 4 and 5).Lastly, rHSA fusion proteins retained stable FA-binding ability, acritical factor for their eventual application as a carrier for FA-Drugs(FIG. 6A). Once forming a complex, rHSA and FA-modified moleculesremained stable even in the presence of excess competing free FA (FIG.6B), as well as serum (FIG. 6C).

Improving serum half-life and retaining target protein binding abilityis only one of the hurdles that must be overcome for a carrier tosuccessfully deliver functional peptides to a cancer cell. In addition,a drug complex must reach the tumor microenvironment and intracellularuptake must occur to allow for target protein interaction and subsequenttherapeutic effect. An important feature of HSA is its ability to crossvascular endothelial cells through albumin-mediated transcytosis andaccumulate in the interstitial space of tumor tissues, a process knownas the enhanced permeability and retention (EPR) effect [39].Intracellular uptake of HSA also occurs under conditions of cellularstress, where it serves as a vital source of amino acids. For instance,it has been reported that tumor cells often have an increased rate ofHSA uptake [21]. While the precise mode of cellular entry is notentirely clear, studies performed to examine the cellular uptake ofAbraxane have revealed that transcytosis is initiated upon binding ofHSA to a cell surface glycoprotein (gp60) receptor (albondin), as wellas binding of HSA to SPARC (secreted protein acid and rich in cysteine)[40]. Apart from receptor-mediated uptake, fluid phase endocytosis isalso suggested to play a role [18]. While future studies will be neededto determine the precise mechanism of rHSA-P53i and rHSA-PMIintracellular uptake, the studies presented here were designed toconfirm that rHSA fusion proteins can be efficiently taken up by cancercells.

To examine cellular uptake, rHSA, rHSA-P53i and rHSA-PMI werefluorescently labeled with FITC. Studies were also performed usingFA-FITC and rHSA complexes to examine whether or not this formulationinterferes with FA-FITC internalization or rHSA fusion protein activity.Following 24-hour incubation with SJSA-1 cells, confocal microscopy wasperformed to determine the extent of rHSA fusion protein andrHSA/FA-FITC uptake. Abundant FITC-staining within intracellularvesicles, as depicted in FIG. 2, indicates FITC-labeled rHSA-P53i,rHSA-PMI, and rHSA were readily taken up into cells. Interestingly, thedegree of internalization was most efficient in cells exposed torHSA-P53i and rHSA-PMI. FIG. 7B confirms FA-FITC, of rHSA/FA-FITCcomplexes, are also effectively delivered intracellularly based on theextensive FITC staining pattern, which is similar to that of FA-FITCtreatment alone.

Although the neonatal Fc receptor (FcRn) may contribute to albumininternalization, the exact mechanism underlying the increased uptake ofrHSA fusion proteins relative to rHSA has yet to be determined. FcRn isa major histocompatibility class I (MHCI) molecule involved in therecycling of both IgG and HSA. It prevents intracellular degradation ofprotein and prolongs its serum half-life. Importantly, IgG and HSAproteins that do not bind FcRn are retained within the cell andeventually processed into lysosomes for proteolytic degradation. Workperformed by Andersen et al. demonstrated that an intact domain III,which contains the C-terminal of HSA, is crucial for FcRn binding andsubsequent exporting back to the cell surface [41]. Future studies willbe necessary to determine if the C-terminal modification of HSAinterferes with FcRn binding, thus promoting intracellular retention ofrHSA fusion proteins.

The overall goal of this technology is to efficiently deliver atherapeutic peptide and potentially, for co-delivery of FA-Drugs toinduce a synergistic response. Thus, we next designed experiments toexamine the cytotoxic effect of rHSA-P53i and rHSA-PMI. Our data revealrHSA-P53i and rHSA-PMI, as well as rHSA-P53i/FA-FITC andrHSA-PMI/FA-FITC complexes, caused significant cytotoxicity in SJSA-1cells (FIGS. 4A and 7D, respectively). In addition, robust caspaseactivation was triggered following rHSA fusion protein treatment. Thisimplies toxicity was related to apoptotic mechanisms (FIG. 4B).

To further elucidate the mechanisms underlying the cytotoxic effects ofrHSA-P53i and rHSA-PMI, Western blots were performed to examine changesin p53 and MDM2. It has been reported that disruption of p53-MDM2binding can prevent the sequestration and subsequent ubiquitination ofp53 by MDM2 and result in accumulation and reactivation of p53. In linewith this mechanism, we observed an increase in p53 protein following a24-hour incubation with rHSA-P53i, rHSA-PMI or nutlin. Unlike nutlin,treatment with rHSA-P53i or rHSA-PMI did not cause an increase in MDM2.

The consequences of p53 activation are highly complex and can bedifferent depending on a number of factors, such as differences instimuli, external environment or cellular milieu. p53-dependent cellularoutcomes are dictated by a myriad of transcriptional targets. It hasbeen shown that p53 transcription stimulates the synthesis of MDM2.However, MDM2 can inhibit the transcriptional activity of p53 by bindingto its transactivation domain. Furthermore, MDM2 can regulate thedegradation of p53 by acting as a shuttle to transport p53 out of thenucleus into the cytosol. Thus, p53 and MDM2 form an autoregulatoryfeedback loop [34-36]. The dynamic regulatory pathway of MDM2 makes ithard to predict the protein expression outcome caused by p53 activation.In a closed system, it could be assumed that p53 accumulation would leadto an increase in MDM2 protein expression. However, in a cellularcontext, one must consider that a number of other factors can affectMDM2 stability and activity. As depicted in FIG. 5, an increase in p53protein expression following treatment with rHSA-P53i and rHSA-PMI wasobserved, while MDM2 remained at basal levels. These results are incontrast to previous studies of MDM2 small molecule antagonists, whichobserved a concomitant increase in MDM2 upon p53 accumulation [10]. Thisinconsistency poses an important question: what levels of MDM2inhibition and p53 activity are required to invoke a beneficialtherapeutic effect? To answer this question, work performed by Mendrysaet al. using mouse models with a hypomorphic allele of MDM2, found thateven small reductions in MDM2 levels were sufficient to cause a mild p53response [42]. Based on these studies, our data may suggest p53activation was beneath the threshold required for promoting p53-mediatedtranscription of MDM2. A second scenario may also exist in whichliberated p53, at different concentrations, triggers transcription of adifferent subset of genes involved in p53-mediated apoptosis that doesnot include MDM2. Lastly, we have considered a transcription-independentapoptotic mechanism mediated by cytoplasmic p53 [43]. It was shown thatp53-dependent apoptosis still occurred in the presence oftranscriptional or translational inhibitors. Furthermore, p53 mutantslacking transcriptional activity retained the ability to triggerapoptotic function. It is possible rHSA-P53i and rHSA-PMI interfere withthe transportation of cytoplasmic p53 into the nucleus. Clearly, furtherstudies will be needed to determine whether or not atranscriptional-independent or -dependent apoptotic pathway underliesrHSA fusion protein cytotoxicity, as well as the exact mechanismsunderlying the maintenance of MDM2 levels following rHSA fusion proteintreatment.

We further explored the potential of using rHSA-P53i or rHSA-PMI as avehicle to co-deliver a FA-Drug as well as a therapeutic peptide. Forease of quantitation and detection, FITC was chosen as the modelmolecule to test the feasibility and stability of this co-deliverytechnology. Recombinant HSA-mediated delivery of FA-Drugs offers anumber of advantages over traditional drug delivery methods. Theseinclude: 1) HSA association improves solubility of FA-Drugs as well asextremely hydrophobic drugs, 2) formation of FA-Drug and rHSA complexoccurs naturally upon incubation and does not require an elaboratechemical reaction, 3) the non-covalent nature of FA-Drug and rHSAnegates the need for protein degradation as in polymer/protein-drugconjugates, and 4) drug absorption could also be enhanced due toincreased hydrophobicity of the fusion polypeptides and HSA-mediateduptake. Generally, LCFAs dissociate from HSA, translocate across thecell membrane, and then reach the mitochondrial membrane and otherintracellular locations. The translocation of LCFA across the cellmembrane may go through two co-existing mechanisms: simple diffusion andsaturable receptor-mediated processes. If a FA-Drug follows the route ofLCFA, it may reach the cytoplasmic target through 1) FAtransporter-mediated translocation or 2) passive diffusion facilitatedby the FA lipophilic alkyl chain. If HSA is involved in thetransportation, FA-Drug may translocate across the cell membranethrough 1) HSA binding protein-mediated endocytosis or 2) increased HSAuptake in tumor cells [21,40]. Thus, multiple uptake pathways may leadto more efficient drug absorption.

As formulation of rHSA/FA-Drug will be performed in vitro, assessing thestability of this complex under simulated in vivo conditions wasnecessary to determine whether significant displacement of FA-Drug fromrHSA complexes will occur in the presence of free fatty acids or serum.The work presented here reveals minimal exchange of FA-FITC took placeeven in the presence of 8 times excess of free FA (FIG. 6B). Inaddition, pre-formulated biotin-rHSA/FA-FITC complexes remained stablefor up to 24 hours in the presence of 10% serum, a condition designed tomimic an in vivo setting (FIG. 6C).

In recent years, many studies have confirmed that blocking p53-MDM2interaction holds promise in reestablishing the p53 tumor suppressorpathway when wild type p53 is present. This is particularly relevant interms of treatment, given that certain cancer cells overexpress MDM2 orMDMX, an MDM2 homolog that also binds and sequesters p53. Structuralstudies of the p53-binding groove within MDM2 led to the development ofboth small molecule peptide mimetics (such as nutlins) and rationallydesigned peptide inhibitors. While nutlins have allowed the mechanisticproof-of-concept for disrupting p53-MDM2 binding for cancer therapy,their pharmacological properties have prevented translation into aclinical model. Peptide inhibitors have the advantage of offering a highdegree of target specificity, as well as the ability in some cases tobind and inhibit both MDM2 and MDMX. Despite this potency, however,peptide inhibitors have demonstrated only modest effects in invitro cellmodels, presumably due to poor membrane permeability and structuralstability. Here we present a method that may not only overcome thecurrent obstacles associated with peptide drug delivery into cells, butalso facilitates the co-transport of small molecule anticancer agents.Although fatty acid modification may enhance the cellular uptake ofmolecules, future studies may be needed to optimize the FA conjugationlinker to promote maximum internalization and cytotoxic activity ofsmall molecule drug candidates.

Example 10

The compositions of this invention are useful for enhancing cellularresponse to apoptosis, for use in cancer therapy, such as cancercombination therapy. p53 protein is a critical cancer suppressor thatsenses intrinsic cellular stresses and controls apoptosis. MDM2 isoverexpressed in many cancer cells, and binds to p53, promoting thedegradation of p53. Disruption of p53 and MDM2 interaction by peptidesand small molecules can to stimulate the accumulation of cellular p53and activate p53-mediated apoptosis, and subsequently sensitize cellularresponse to chemotherapeutics. There are two major biological functionsof wild type p53, transcription-dependent and independent cellularregulations. In addition to the transcription functions of cellular p53,p53 can directly bind and inhibit two anti-apoptotic proteins, BCL-XLand MCL and induce apoptosis. No studies have shown that p53 can induceapoptosis in p53 mutant cancer cells such as p53 negative cells or cellsunderexpressing p53 (including cells expressing p53 with lower apoptoticmediating activity than wild-type p53. It has been demonstrated hereinthat p53-derived peptides (that do not include active p53) cansurprisingly bind and interact with four targets, MDM2, MDMX, BCL-XL,and MCL and induce cytotoxicity independent on p53 genotype (wild type,or mutant, including p53 negative or p53 underexpressing cells). Thisfinding expands the application of p53-derived peptides and analogues tomost cancer cells, even p53 negative or p53 underexpressing cells.Previously, no small molecules targeting apoptosis mechanism have beenshown can inhibit more than two targets involved in mediating apoptosisefficiently.

Example 11 Exemplary Fusion Polypeptide Binding to BCL-XL and MCL-1 (forExample, rHSA-P53i and rHSA-PMI)

As shown herein, rHSA-P53i and rHSA-PMI induce apoptosis in SJSA-1 cells(p53 positive cancer cell line). This is explained by the fact that p53functions as a tumor suppressor by promoting the expression ofpro-apoptotic proteins capable of triggering apoptosis via caspaseactivation. However, it has also been surprisingly found, that exemplaryfusion polypeptides, rHSA-P53i and rHSA-PMI, induce apoptosis in othercell lines, including MDA-MB-231 (a p53 negative cancer cell line), orHela (a p53 under-expressing cancer cell line).

To elucidate the molecular basis for the above surprising observation,the inventors explored whether proteins like Bcl-xL/Mcl-1 interact withrHSA-P53i and rHSA-PMI. We added biotin-rHSA, biotin-rHSA-P53i, orbiotin-rHSA-PMI to MDA-MB-231 (p53 negative cancer cell line), SJSA-1(p53 positive cancer cell line) or Hela (p53 under-expressing cancercell line) whole cell lysates. Proteins were then pulled down usinganti-Bcl-xL antibody, anti-Mcl-1 antibody or streptavidin and followedby Western blotting using streptavidin-HRP (to detect biotinylatedrHSA), anti-Bcl-xL or anti-Mcl-1 antibodies. The results are shown inFIG. 9. As depicted in FIG. 9A, anti-Bcl-xL antibody pulled downbiotin-rHSA-P53i (lane 2) and biotin-rHSA-PMI (lane 3) but notbiotin-rHSA (lane 1), from MDA-MB-231 (a), SJSA-1 (b) or Hela (c) wholecell lysates. Similarly, as shown in FIG. 9B, anti-Mcl-1 antibody pulleddown biotin-rHSA-P53i (lane 2) and biotin-rHSA-PMI (lane 3) but notbiotin-rHSA (lane 1), from MDA-MB-231 (a), SJSA-1 (b) or Hela (c) wholecell lysates. As illustrated in FIG. 9C, streptavidin pulled down Bcl-xLand Mcl-1, when biotin-rHSA-P53i or biotin-rHSA-PMI (lane 2) but notwhen HSA (lane 1) was added. Again these protein were pulled down fromMDA-MB-231, SJSA-1 as well as Hela whole cell lysates. These datademonstrate that the exemplary fusion polypeptides, rHSA-P53i andrHSA-PMI, surprisingly bind to Bcl-xL and Mcl-1.

Example 12 Exemplary Fusion Polypeptide rHSA-P53i Reduces Bak-Bcl-xL andBAK-MCL-1 Interactions

Having shown that rHSA-P53i and rHSA-PMI bind to Bcl-xL and Mcl-1, itwas next determined whether rHSA fusion proteins were able to displaceBak from the BH3 binding groove of Bcl-xL. Towards that biotin-rHSA,biotin-rHSA-P53i, or biotin-rHSA-PMI was added to MDA-MB-231 (p53negative cancer cell line), SJSA-1 (p53 positive cancer cell line) orHela (p53 under-expressing cancer cell line) whole cell lysates.Proteins were then pulled down using anti-Bcl-xL antibody and sampleswere then analyzed by SDS-PAGE and Western blotting using Bcl-xL, Bak,and Streptavidin-HRP (Strep-HRP) antibodies. The data for SJSA-1, Helaand MDA-MB-231 are depicted in FIGS. 10A, 10B, and 10C, respectively.The amount of protein in each band was determined using Image J software(data not shown). Quantitation of band intensities showed thatbiotin-rHSA-P53i and biotin-rHSA-PMI reduced Bak-Bcl-xL interaction(data not shown).

In a separate experiment, biotin-rHSA or biotin-rHSA-P53i was added toMDA-MB-231, SJSA-1 or Hela whole cell lysates. Proteins were then pulleddown using anti-MCL-1 antibody and samples were then analyzed bySDS-PAGE and Western blotting using MCL-1, BAK, and Streptavidin-HRP(Strep-HRP) antibodies. The data, exhibited in FIG. 10D, shows that theamount of BAK pulled down by anti-MCL-1 antibody was lesser whenbiotin-rHSA-P53i was added to cell lysate, compared to when biotin-rHSAwas added. The amount of protein in each band was determined using ImageJ software (data not shown). Quantitation of band intensities showedthat rHSA-P53i reduced MCL-1 interaction (data not shown). It is likelythat biotin-rHSA-P53i and biotin-rHSA-PMI reduce interaction betweenother pro-apoptotic and anti-apoptotic Bcl-2 family members.

Example 13 Exemplary Fusion Polypeptide rHSA-P53i Promotes Release ofCytochrome C

It is well known that Bcl-XL, MCL-1 and BAK are Bcl-2 family members.There are a total of 25 genes in the Bcl-2 family known to date, whichare classified in to either pro-apoptotic (Bax, Diva, BCl-Xs, Bik, Bim,Bad, Bid, Bak, Bok, Egl-1, Bax, etc) or anti-apoptotic (including Bcl-2proper, Bcl-xL, and Bcl-w, CED-9, A1, Bfl-1, among an assortment ofothers) members. These proteins govern mitochondrial outer membranepermeabilization. Disruption of mitochondrial outer membranepermeabilization leads to release of cytochrome C into the cytosolwhich, once there, activates caspase-9 and caspase-3, leading toapoptosis. It is thought action of the pro-apoptotic members of Bcl-2family proteins induces, and anti-apoptotic members inhibits themitochondrial outer membrane permeabilization.

Since the exemplary fusion polypeptide, rHSA-P53i, reduces Bak-Bcl-xLand BAK-MCL-1 interactions, it was next determined how the fusionpolypeptide affects apoptosis in target cancer cells having differentp53 genotypes. Towards that, increasing amounts of rHSA-P53i were addedto Hela, SJSA-1 or MDA-MB-231 cells. Cytosolic and mitochondrialfractions were isolated and subjected to Western blotting usinganti-Cytochrome C antibody. As shown in FIG. 11, the amount ofcytochrome C increased in the cytoplasmic fraction in a dose dependentmanner, in all three cell lines, irrespective of their p53 status. Therewas a corresponding decrease in the amount of cytochrome C in themitochondrial fraction. The release cytochrome C is expected to oncethere, activates caspase-9 and caspase-3, leading to apoptosis. Thesedata show that rHSA-P53i promotes release of cytochrome C, irrespectiveof p53 genotype, and phenotype of the cancer cells.

Example 14 Exemplary Fusion Polypeptide rHSA-P53i Co-Localizes withMitochondria in SJSA-1 and Hela Cells

In order to determine whether rHSA-P53i co-localizes with mitochondria,SJSA-1 and Hela cells were treated with FITC-labeled rHSA,FITC-rHSA-P53i, and FITC-rHSA-PMI. Mitochondrial and nuclear stainingwas performed using MitoTracker Deep Red (red) and Hoechst 33342 (blue),and the cells were then subjected to immunofluorescence microscopy. Asshown in FIGS. 12A and C, HSA showed punctate localization. This may beconsistent with localization of serum albumin with lysosomal system orsecretory apparatus as has been previously seen (Yokota and Fahimi,Proceedings of National Academy of Sciences of the United States ofAmerica, 78: 4970-4974, 1981; Baghdiguian et al., Cancer Letters101179-84, 1996). Despite efficient rHSA uptake in all conditions,mitochondrial co-localization was not observed in cells treated withrHSA (A and C). In contrast, visualization at 60× magnification revealedabundant yellow staining in cells treated with rHSA-P53i (B and D), andrHSA-PMI (data not shown) indicating the exemplary fusion polypeptide,rHSA-P53i, efficiently co-localized with mitochondrial organelles.

Example 15 Cytotoxic Activity of Exemplary Fusion Polypeptides,rHSA-P53i and rHSA-PMI, is Independent of p53 Status

To determine whether rHSA-P53i and rHSA-PMI induce to cell death, HSA orthe rHSA fusion proteins were added to MDA-MB-231, SJSA-1 or Hela cellsand allowed to incubate for 24 hrs. Nutlin was used as a negativecontrol in MDA-MB-231 (p53-mutant) and Hela (unstable wild type p53)cells as it relies on a wild type p53-dependent cytotoxic mechanism, andABT-263, a BH3 mimetic (Bcl-xL inhibitor), was included as a positivecontrol to confirm the presence of functional mitochondrial-mediatedcytotoxic pathways. Cytotoxicities were measured by CyQuant Assay andnormalized according to rHSA-treated cells. A bar graph showing celldeath, relative to rHSA-treated cells, from representative of threeindependent experiments, performed in triplicate, are displayed in FIG.13. As shown, nutlin killed p53-positive SJSA-1 cells, but notMDA-MB-231 or Hela cells, showing requirement for wild type levels ofwild type p53 in the mechanism of cell death by nutlin. ABT-263 (ABT)killed MDA-MB-231 and Hela cells showing presence ofmitochondrial-mediated cytotoxic pathways. Both rHSA-P53i and rHSA-PMIinduced cell death in SJSA-1 cells (p53 positive cancer cell line) aswell as MDA-MB-231 cells (p53 negative cancer cell line), or Hela cells(p53 under-expressing cancer cell line), illustrating cytotoxic activityof exemplary fusion polypeptides rHSA-P53i and rHSA-PMI is independentof p53 status.

Example 16 Co-Administration of an Exemplary Fusion Polypeptide,rHSA-PMI, and an Exemplary Anticancer Agent, Methotrexate (MTX),Enhances Apoptotis Compared to the Agent Alone in SJSA-1 Cells

As shown above, HSA-PMI may induce apoptosis by disrupting MDM2/MDMX-p53interaction and/or by inducing the release of BAK from MCL-1 or BCL-X1.This mechanism may suggest that the agent may show synergistic effectwith many anticancer agents. To test this hypothesis, SJSA cells weretreated with MTX alone, HSA-PMI/nutlin alone, or MTX and HSA-PMI/nutlin.Nutlin was used as an example of as shown in FIG. 14A, the amount ofcell death induced by MTX was not additive with that induced by nutlin;however, the amount of cell death induced by MTX and HSA-PMI wasenhanced by each other. This demonstrates that the fusion polypeptidesof this invention provide surprisingly synergistic results whenco-administered with an anti-cancer agent.

Example 17 Exemplary Fusion Polypeptide, HSA-PMI, has a SynergisticEffect on SJSA-1 Xenograft Tumor Model

To extend the above findings to in vivo settings, SJSA-1 xenografts weretreated with HSA, HSA/MTX, HSA/FA-MTX, and an exemplary fusionpolypeptide, rHSA-PMI/MTX, as indicated in FIG. 14. These data show thatefficacy of HSA/FA-MTX was enhanced by PMA as evidenced by rHSA-PMI/MTXtreatment.

To determine whether exemplary fusion polypeptides, rHSA-P53i andrHSA-PMI, induce cell death, HSA or the rHSA fusion proteins were addedto MDA-MB-231, SJSA-1 or Hela cells and allowed to incubate for 24 hrs.Nutlin was used as a negative control in MDA-MB-231 (p53-mutant) andHela (unstable wild type p53) cells as it relies on a wild typep53-dependent cytotoxic mechanism, and ABT-263, a BH3 mimetic (Bcl-xLinhibitor), was included as a positive control to confirm the presenceof functional mitochondrial-mediated cytotoxic pathways. Cytotoxicitieswere measured by CyQuant Assay and normalized according to rHSA-treatedcells. A bar graph showing cell death, relative to rHSA-treated cells,from representative of three independent experiments, performed intriplicate, are displayed in FIG. 13. As shown, nutlin killedp53-positive SJSA-1 cells, but not MDA-MB-231 or Hela cells, showingrequirement for wild type levels of wild type p53 in the mechanism ofcell death by nutlin. ABT-263 (ABT) killed MDA-MB-231 and Hela cellsshowing presence of mitochondrial-mediated cytotoxic pathways. BothrHSA-P53i and rHSA-PMI induced cell death in SJSA-1 cells (p53 positivecancer cell line) as well as MDA-MB-231 cells (p53 negative cancer cellline), or Hela cells (p53 under-expressing cancer cell line),illustrating that the cytotoxic activity of exemplary fusionpolypeptides of this invention, rHSA-P53i and rHSA-PMI, is surprisinglyindependent of p53 status.

The methods and compositions of this invention are therefore useful indeveloping new therapeutic approaches to cancer. Restoring p53 functionis a common approach used in cancer therapy, but that approach requiredeither that the cancer cell has functional p53 or delivery of extrinsicfunctional p53 into cancer cells. These approaches were focused on therestoring of p53 function by blocking MDM2 and p53 interaction andaccordingly, p53-derived peptides or analogues have not previously beentested for induction of apoptosis in p53 deletion or mutant cancers. Asshown above, p53-derived peptide or p53-activating peptides provided inthe fusion polypeptides of this invention can induce cytotoxicityregardless of functional p53 or p53 genotype. Previous studies haveshown that p53 can interact with BCL-XL and MCL, but the relevance ofthose interactions was not clear since no studies previouslydemonstrated the cytotoxicity of p53-peptides such as a p53-derivedpeptide or p53-activating peptide in cells with p53 mutations, includingp53 negative cells or cells expressing low activities of p53. Theresults of the study set forth herein proves that the fusionpolypeptides of this invention can effectively kill cancer cellsindependent of the presence of functional p53 or p53 genotype orphenotype. The finding that p53-derived peptide or p53-activatingpeptides efficiently induce apoptosis in all cell lines tested indicatesthat the fusion polypeptides of this invention have broad applicationfor delivery of p53 agonists, such as p53-peptides, for example,p53-derived peptides and p53-activating peptides or their analogs fortreatment of most cancers.

This is the first demonstration of applying a therapy previouslyregarded as “p53-dependent” to cells, regardless of p53 genotype, andtherefore, it surprisingly expands the therapeutic spectrum ofp53-derived peptides, p53-activating peptides and/or their peptide orpeptidomitice or small molecule analogs for use in any of the methods ofthis invention. There are other approaches to target either BCL-XL orMCL to induce apoptosis, but not both, and cancer cells have been shownto quickly develop resistance to approaches to target either BCL-XL orMCL. There are approaches to target MDM2 or MDMX, but these approachesdo not tackle BCL-XL or MCL, or have never been tried in cells with p53mutation or deletion. The proposed approach can achieve the goal: onestone, four birds for any direction (theoretically, most cancer cells).

The strategy of using p53-derived peptide or p53-activating peptides maybe combined with an efficient peptide delivery strategy. Further, thep53-derived peptide or p53-activating peptides can be administered incombination with chemotherapeutics to boost synergistic efficacy. Thepower of human serum albumins to carry other drugs allows one tosimultaneously administer molecule binds to multiple therapeutic agents,for example administering agents modulating four targets and affects twoessential pathways. This approach will allow countering the ability ofcancer cells to develop resistance.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims. For example, any of the fusion polypeptides described herein maybe used in any of the methods described herein, or as exemplified in anyof the examples.

REFERENCES

-   1. Zhang X X, Eden H S, Chen X (2012) Peptides in cancer    nanomedicine: drug carriers, targeting ligands and protease    substrates. J Control Release 159: 2-13.-   2. Hupp T R, Meek D W, Midgley C A, Lane D P (1992) Regulation of    the specific DNA binding function of p53. Cell 71: 875-886.-   3. el-Deiry W S (1998) Regulation of p53 downstream genes. Semin    Cancer Biol 8: 345-357.-   4. Ventura A, Kirsch D G, McLaughlin M E, Tuveson D A, Grimm J et    al. (2007) Restoration of p53 function leads to tumour regression in    vivo. Nature 445: 661-665.-   5. Rinn J L, Huarte M. (2011) To repress or not to repress: this is    the guardian's question. Trends Cell Biol 21: 344-353.-   6. Bond G L, Hu W, Levine A J (2005) MDM2 is a central node in the    p53 pathway: 12 years and counting. Curr Cancer Drug Targets 5: 3-8.-   7. Freedman D A, Wu L, Levine A J (1999) Functions of the MDM2    oncoprotein. Cell Mol Life Sci 55: 96-107.-   8. Bottger A, Bottger V, Garcia-Echeverria C, Chene P, Hochkeppel H    K et al. (1997) Molecular characterization of the hdm2-p53    interaction. J Mol Biol 269: 744-756.-   9. Yang Y, Ludwig R L, Jensen J P, Pierre S A, Medaglia M V et    al. (2005) Small molecule inhibitors of HDM2 ubiquitin ligase    activity stabilize and activate p53 in cells. Cancer Cell 7:    547-559.-   10. Vassilev L T, Vu B T, Graves B, Carvajal D, Podlaski F et    al. (2004) In vivo activation of the p53 pathway by small-molecule    antagonists of MDM2. Science 303: 844-848.-   11. Hu B, Gilkes D M, Farooqi B, Sebti S M, Chen J (2006) MDMX    overexpression prevents p53 activation by the MDM2 inhibitor Nutlin.    J Biol Chem 281: 33030-33035.-   12. Wade M, Wong E T, Tang M, Stommel J M, Wahl G M (2006) Hdmx    modulates the outcome of p53 activation in human tumor cells. J Biol    Chem 281: 33036-33044.-   13. Brown C J, Lain S, Verma C S, Fersht A R, Lane D P (2009)    Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer    9: 862-873.-   14. Li C, Pazgier M, Yuan W, Liu M, Wei G et al. (2010) Systematic    mutational analysis of peptide inhibition of the p53-MDM2/MDMX    interactions. J Mol Biol 398: 200-213.-   15. Pazgier M, Liu M, Zou G, Yuan W, Li C et al. (2009) Structural    basis for high-affinity peptide inhibition of p53 interactions with    MDM2 and MDMX. Proc Natl Acad Sci USA 106: 4665-4670.-   16. Fasano M, Curry S, Terreno E, Galliano M, Fanali G et al. (2005)    The extraordinary ligand binding properties of human serum albumin.    IUBMB Life 57: 787-796.-   17. Chuang V T, Kragh-Hansen U, Otagiri M (2002) Pharmaceutical    strategies utilizing recombinant human serum albumin Pharm Res 19:    569-577.-   18. Kratz F (2008) Albumin as a drug carrier: design of prodrugs,    drug conjugates and nanoparticles. J Control Release 132: 171-183.-   19. Gradishar W J, Tjulandin S, Davidson N, Shaw H, Desai N et    al. (2005) Phase III trial of nanoparticle albumin-bound paclitaxel    compared with polyethylated castor oil-based paclitaxel in women    with breast cancer. J Clin Oncol 23: 7794-7803.-   20. Vousden K H, Prives C (2009) Blinded by the Light: The Growing    Complexity of p53. Cell 137: 413-431.-   21. Boratyński J, Opolski A, Wietrzyk J, Gorski A, Radzikowski    C (2000) Cytotoxic and antitumor effect of fibrinogen-methotrexate    conjugate. Cancer Lett 148: 189-195.-   22. Tzefos M, Olin J L Glucagon-like peptide-1 analog and insulin    combination therapy in the management of adults with type 2 diabetes    mellitus. Ann Pharmacother 44: 1294-1300.-   23. Peterson G E (2006) Intermediate and long-acting insulins: a    review of NPH insulin, insulin glargine and insulin detemir. Curr    Med Res Opin 22: 2613-2619.-   24. Rustgi V K (2009) Albinterferon alfa-2b, a novel fusion protein    of human albumin and human interferon alfa-2b, for chronic    hepatitis C. Curr Med Res Opin 25: 991-1002.-   25. Pignatello R, Guccione S, Forte S, Di Giacomo C, Sorrenti V et    al. (2004) Lipophilic conjugates of methotrexate with short-chain    alkylamino acids as DHFR inhibitors. Synthesis, biological    evaluation, and molecular modeling. Bioorg Med Chem 12: 2951-2964.-   26. Singh Y, Palombo M, Sinko P J (2008) Recent trends in targeted    anticancer prodrug and conjugate design. Curr Med Chem 15:    1802-1826.-   27. Kratz F, Müller I A, Ryppa C, Warnecke A (2008) Prodrug    strategies in anticancer chemotherapy. Chemmedchem 3: 20-53.-   28. Cregg J M, Tolstorukov I, Kusari A, Sunga J, Madden K et    al. (2009) Expression in the yeast Pichia pastoris. Methods Enzymol    463: 169-189.-   29. Travis J, Bowen J, Tewksbury D, Johnson D, Pannell R (1976)    Isolation of albumin from whole human plasma and fractionation of    albumin-depleted plasma. Biochem J 157: 301-306.-   30. Chen R F (1967) Removal of fatty acids from serum albumin by    charcoal treatment. J Biol Chem 242: 173-181.-   31. Kussie P H, Gorina S, Marechal V, Elenbaas B, Moreau Jet    al. (1996) Structure of the MDM2 oncoprotein bound to the p53 tumor    suppressor transactivation domain. Science 274: 948-953.-   32. Schon O, Friedler A, Bycroft M, Freund S M, Fersht A R (2002)    Molecular mechanism of the interaction between MDM2 and p53. J Mol    Biol 323: 491-501.-   33. El-Deiry W S (2003) The role of p53 in chemosensitivity and    radiosensitivity. Oncogene 22: 7486-7495.-   34. Weber J D, Jeffers J R, Rehg J E, Randle D H, Lozano G et    al. (2000) p53-independent functions of the p19(ARF) tumor    suppressor. Genes Dev 14: 2358-2365.-   35. Tao W, Levine A J (1999) P19(ARF) stabilizes p53 by blocking    nucleo-cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci USA 96:    6937-6941.-   36. Kuo M L, Duncavage E J, Mathew R, den Besten W, Pei D et    al. (2003) Arf induces p53-dependent and -independent    antiproliferative genes. Cancer Res 63: 1046-1053.-   37. Barak Y, Juven T, Haffner R, Oren M (1993) Mdm2 expression is    induced by wild type p53 activity. EMBO J 12: 461-468.-   38. Spector A A (1975) Fatty acid binding to plasma albumin J Lipid    Res 16: 165-179.-   39. John T A, Vogel S M, Tiruppathi C, Malik A B, Minshall R    D (2003) Quantitative analysis of albumin uptake and transport in    the rat microvessel endothelial monolayer. Am J Physiol Lung Cell    Mol Physiol 284: L187-L196.-   40. Desai N, Trieu V, Yao Z, Louie L, Ci S, et al. (2006) Increased    antitumor activity, intratumor paclitaxel concentrations, and    endothelial cell transport of cremophor-free, albumin-bound    paclitaxel, ABI-007, compared with cremophor-based paclitaxel. Clin    Cancer Res 12: 1317-1324.-   41. Andersen J T, Dalhus B, Cameron J, Daba M B, Plumridge A et    al. (2012) Structure-based mutagenesis reveals the albumin-binding    site of the neonatal Fc receptor. Nat Commun 3: 610.-   42. Mendrysa S M, McElwee M K, Michalowski J, O'Leary K A, Young K M    et al. (2003) Mdm2 Is critical for inhibition of p53 during    lymphopoiesis and the response to ionizing irradiation. Mol Cell    Biol 23: 462-472.-   43. Caelles C, Helmberg A, Karin M (1994) p53-dependent apoptosis in    the absence of transcriptional activation of p53 target genes.    Nature 370: 220-223.

1.-15. (canceled)
 16. A method of disrupting BCL-XL and MCL-1 inhibitionof apoptosis in a cell, the method comprising: (a) providing a fusionpolypeptide; and (b) contacting the cell with the fusion polypeptide,thereby disrupting BCL-XL and MCL-1 inhibition of apoptosis in the cell,wherein the fusion polypeptide comprises a human serum albumin and ap53-peptide, further comprising one or more anticancer agent, whereinthe one or more anticancer agent is bound to the human serum albumin viacovalent interactions, or is chemically conjugated to a natural ligandof the human serum albumin, wherein the natural ligand is bound to thehuman serum albumin.
 17. The method of claim 16, wherein the cancer cellis selected from the group consisting of: (a) a p53-wild-type cancercell; (b) a p53 mutant cancer cell; or (c) a p53-negative cancer cell.18. The method of claim 16, wherein the cancer is selected from thegroup consisting of Acute Lymphoblastic Leukemia (ALL), acute myeloidleukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-relatedlymphoma, anal cancer, appendix cancer, astrocytoma, childhoodcerebellar or cerebral cancer, basal-cell carcinoma, bile duct cancer,bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma,brainstem glioma, brain cancer, brain tumor, cerebellar astrocytoma,cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,Burkitt's lymphoma, carcinoid tumor, childhood tumor, carcinoid tumor,gastrointestinal, carcinoma of unknown primary, central nervous systemlymphoma, primary, childhood cerebellar astrocytoma, childhood cerebralastrocytoma/malignant glioma, cervical cancer, cholangiocarcinoma,chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, desmoplastic small round cell tumor, endometrialcancer, ependymoma, esophageal cancer, ewing's sarcoma in the ewingfamily of tumors, childhood extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, intraocular melanoma,eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach)cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor(GIST), germ cell tumor, extracranial germ cell tumor, extragonadal germcell tumor, ovarian germ cell tumor, gestational trophoblastic tumor,glioma of the brain stem, glioma, childhood cerebral astrocytoma,glioma, visual pathway and hypothalamic cancer, gastric carcinoidcancer, hairy cell leukemia, head and neck cancer, heart cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, non-Hodgkin lymphoma,hypopharyngeal cancer, hypothalamic and visual pathway glioma,intraocular melanoma, islet cell carcinoma (e.g. endocrine, pancreatic),Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer,leukaemia, acute lymphoblastic leukaemia (also called acute lymphocyticleukaemia), acute myeloid leukaemia (also called acute myelogenousleukemia), chronic lymphocytic leukaemia (also called chroniclymphocytic leukemia), chronic myelogenous leukemia (also called chronicmyeloid leukemia), hairy cell leukemia, lip and oral cavity cancer,liposarcoma, liver cancer (primary), lung cancer, non-small cell lungcancer, small cell lung cancer, AIDS-related lymphoma, Burkitt lymphoma,cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (anold classification of all lymphomas except Hodgkin's), primary centralnervous system cancer, macroglobulinemia, Waldenström, male breastcancer, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanoma, melanoma, intraocular (eye) cancer, merkelcell cancer, mesothelioma, adult malignant mesothelioma, metastaticsquamous neck cancer with occult primary, mouth cancer, multipleendocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm,mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, myelogenous leukemia,chronic, myeloid leukemia, adult acute, myeloid leukemia, childhoodacute myeloma, multiple myeloma, chronic myeloproliferative disorder,myxoma, nasal cavity and paranasal sinus cancer, nasopharyngealcarcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma,oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibroushistiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surfaceepithelial-stromal tumor), ovarian germ cell tumor, ovarian lowmalignant potential tumor, pancreatic cancer, pancreatic islet cellcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,pineal germinoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary adenoma, plasma cellneoplasia/multiple myeloma, pleuropulmonary blastoma, primary centralnervous system lymphoma, prostate cancer, rectal cancer, renal cellcarcinoma (kidney cancer), renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma, ewing family of tumors, sarcoma, kaposi, sarcoma, soft tissue,sarcoma, uterine, sézary syndrome, skin cancer (non-melanoma), skincancer (melanoma), skin carcinoma, merkel cell, small cell lung cancer,small intestine cancer, soft tissue sarcoma, squamous cell carcinoma,squamous neck cancer with occult primary, metastatic, stomach cancer,supratentorial primitive neuroectodermal tumor, T-cell lymphoma,cutaneous fungoides and sézary syndrome, testicular cancer, throatcancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,trophoblastic tumor, gestational, carcinoma of unknown primary site,cancer of unknown primary site, transitional cell cancer, urethralcancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer,visual pathway and hypothalamic glioma, childhood cancer, vulvar cancer,waldenström macroglobulinemia, and Wilms tumor (kidney cancer). 19.-20.(canceled)
 21. The method of claim 16, wherein the one or moreanticancer agent is chemically conjugated to a natural ligand of thehuman serum albumin.
 22. The method of claim 21, wherein the naturalligand of the human serum albumin is a fatty acid, an amino acid, anutrient, a vitamin, a metabolite, an hormone, or a drug.
 23. The methodof claim 21, wherein the wherein the natural ligand of human serumalbumin is a fatty acid.
 24. The method of claim 16, wherein thep53-peptide is selected from the group consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO:7.
 25. A method of treating asubject with a cancer responsive to p53 inhibition of BCL-XL and MCL-1,and disrupting p-53 MDM2 interactions, the method comprising: (a)administering a therapeutically effective amount of a fusionpolypeptide, wherein the fusion polypeptide comprises a serum albumin,or a fragment thereof, and a p53-peptide, wherein the fragment retainsthe cell transport property, ligand binding property or both celltransport and ligand binding properties.
 26. The method of claim 25,wherein the fusion polypeptide is a recombinant fusion polypeptidecomprising: (a) a p53-derived peptide and a serum albumin polypeptide,or a fragment thereof; or (b) a p53-activating peptide and a serumalbumin, or a fragment thereof.
 27. The method of claim 25, wherein thecancer is selected from the group consisting of: (a) a p53-wild-typecancer cell; (b) a p53 mutant cancer cell; or (c) a p53-negative cancercell.
 28. The method of claim 25, wherein the cancer is selected fromthe group consisting of Acute Lymphoblastic Leukemia (ALL), acutemyeloid leukemia, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma,childhood cerebellar or cerebral cancer, basal-cell carcinoma, bile ductcancer, bladder cancer, bone tumor, osteosarcoma/malignant fibroushistiocytoma, brainstem glioma, brain cancer, brain tumor, cerebellarastrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors, visualpathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor, childhoodtumor, carcinoid tumor, gastrointestinal, carcinoma of unknown primary,central nervous system lymphoma, primary, childhood cerebellarastrocytoma, childhood cerebral astrocytoma/malignant glioma, cervicalcancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic smallround cell tumor, endometrial cancer, ependymoma, esophageal cancer,ewing's sarcoma in the ewing family of tumors, childhood extracranialgerm cell tumor, extragonadal germ cell tumor, extrahepatic bile ductcancer, intraocular melanoma, eye cancer, retinoblastoma, gallbladdercancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), germ cell tumor, extracranialgerm cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor, glioma of the brain stem, glioma,childhood cerebral astrocytoma, glioma, visual pathway and hypothalamiccancer, gastric carcinoid cancer, hairy cell leukemia, head and neckcancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma,non-Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visualpathway glioma, intraocular melanoma, islet cell carcinoma (e.g.endocrine, pancreatic), Kaposi sarcoma, kidney cancer (renal cellcancer), laryngeal cancer, leukaemia, acute lymphoblastic leukaemia(also called acute lymphocytic leukaemia), acute myeloid leukaemia (alsocalled acute myelogenous leukemia), chronic lymphocytic leukaemia (alsocalled chronic lymphocytic leukemia), chronic myelogenous leukemia (alsocalled chronic myeloid leukemia), hairy cell leukemia, lip and oralcavity cancer, liposarcoma, liver cancer (primary), lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma (an old classification of all lymphomas exceptHodgkin's), primary central nervous system cancer, macroglobulinemia,Waldenström, male breast cancer, malignant fibrous histiocytoma ofbone/osteosarcoma, medulloblastoma, melanoma, melanoma, intraocular(eye) cancer, merkel cell cancer, mesothelioma, adult malignantmesothelioma, metastatic squamous neck cancer with occult primary, mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasmacell neoplasm, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, myelogenous leukemia,chronic, myeloid leukemia, adult acute, myeloid leukemia, childhoodacute myeloma, multiple myeloma, chronic myeloproliferative disorder,myxoma, nasal cavity and paranasal sinus cancer, nasopharyngealcarcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma,oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibroushistiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surfaceepithelial-stromal tumor), ovarian germ cell tumor, ovarian lowmalignant potential tumor, pancreatic cancer, pancreatic islet cellcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,pineal germinoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary adenoma, plasma cellneoplasia/multiple myeloma, pleuropulmonary blastoma, primary centralnervous system lymphoma, prostate cancer, rectal cancer, renal cellcarcinoma (kidney cancer), renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoma, ewing family of tumors, sarcoma, kaposi, sarcoma, soft tissue,sarcoma, uterine, sézary syndrome, skin cancer (non-melanoma), skincancer (melanoma), skin carcinoma, merkel cell, small cell lung cancer,small intestine cancer, soft tissue sarcoma, squamous cell carcinoma,squamous neck cancer with occult primary, metastatic, stomach cancer,supratentorial primitive neuroectodermal tumor, T-cell lymphoma,cutaneous fungoides and sézary syndrome, testicular cancer, throatcancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,trophoblastic tumor, gestational, carcinoma of unknown primary site,cancer of unknown primary site, transitional cell cancer, urethralcancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer,visual pathway and hypothalamic glioma, childhood cancer, vulvar cancer,waldenström macroglobulinemia, and Wilms tumor (kidney cancer).
 29. Themethod of claim 25, wherein the fusion polypeptide further comprises asmall molecule drug.
 30. The method of claim 29, wherein the smallmolecule drug is chemically conjugated to a natural ligand of the serumalbumin or the fragment thereof.
 31. The method of claim 29, wherein thesmall molecule drug is bound to a natural ligand of the serum albumin orthe fragment thereof.
 32. The method of claim 31, wherein the naturalligand is a fatty acid, an amino acid, a nutrient, a vitamin, ametabolite, an hormone, or a drug.
 33. The method of claim 31, whereinthe natural ligand of human serum albumin is a fatty acid.
 34. A methodof inducing apoptosis in a cell by one or more of: inhibiting BCL andMCL-1, disrupting p53-MDM2 interaction and disrupting p53-MDMXinteraction, the method comprising: (a) providing a fusion polypeptide;and (b) contacting the cell with the fusion polypeptide, therebyinducing apoptosis in the cell, wherein the fusion polypeptide comprisesa serum albumin, or a fragment thereof, and a p53-peptide, wherein thefragment retains the cell transport property, ligand binding property orboth cell transport and ligand binding properties.
 35. The method ofclaim 34, wherein the cell is selected from the group consisting of: (a)a p53-wild-type cancer cell; (b) a p53 mutant cancer cell; or (c) ap53-negative cancer cell.
 36. The method of claim 34, wherein the fusionpolypeptide further comprises a small molecule drug.
 37. The method ofclaim 36, wherein the small molecule drug is bound to a natural ligandof the serum albumin or the fragment thereof.